Cattle reproductive disease vaccines

ABSTRACT

The present invention relates to combination vaccines and methods for treating or preventing diseases or disorders in an animal caused by infection by Bovine Viral Diarrhea Virus (BVDV) Types 1 and 2, Bovine Herpes Virus Type-1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Virus (PI 3 ),  Campylobacter fetus, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardj - prajitno, Leptospira icterohaemmorrhagiae, Leptospira hardjo - bovis  and  Leptospira pomona  by administering to the animal an effective amount of a combination vaccine. The combination vaccine can be a whole or partial cell inactivated or modified live preparation.

FIELD OF THE INVENTION

[0001] The present invention relates to combination vaccines and methodsfor treating or preventing diseases or disorders in an animal caused byinfection by Bovine Viral Diarrhea Virus (BVDV) Types 1 and 2, BovineHerpes virus Type-1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV),Parainfluenza Virus (PI₃), Campylobacter fetus, Leptospira canicola,Leptospira grippotyphosa, Leptospira borgpetersenii hardjo-prajitno,Leptospira icterohaemmorrhagiae, Leptospira borgpetersenii hardjo-bovisand Leptospira interrogans pomona by administering to the animal aneffective amount of a combination vaccine. The combination vaccine canbe a whole or partial cell inactivated or modified live preparation.

BACKGROUND OF THE INVENTION

[0002] Five viral agents associated with the bovine respiratory disease(BRD) complex—Bovine Herpes Virus Type-1 (BHV-1), also known asinfectious bovine rhinotracheitis virus (IBR), Bovine viral diarrheavirus (BVDV) Types 1 and 2, Bovine Respiratory Syncytial Virus (BRSV),and Parainfluenza Virus (PI₃), cause respiratory and reproductive systeminfections of great economic importance to the cow-calf and dairyindustries worldwide. BRD causes a broad array of clinical syndromesincluding acute onset respiratory disease and abortion. The respiratoryform of BRD is characterized by inflammation, swelling, hemorrhage, andnecrosis of the mucous membranes of the respiratory tract and may beaccompanied by high fever, anorexia, depression, nasal discharge,labored breathing, and inflamed muzzle. Abortions induced by IBR andBVDV virus can occur in all three trimesters, but chiefly during thelast half of gestation, and often without evidence of other clinicalsigns (Ellis et al. (1996) JAVMA 208:393-400; Ellsworth et al. (1994)In: Proceedings, 74^(th) Conference of Research Workers in AnimalDisease:34).

[0003] Bovine Herpes Virus Type-1 (BHV-1), is a member of thealphaherpesviridae subfamily, and produces a variety of clinical formsof disease in cattle, including respiratory and genital infections,conjunctivitis, encephalitis, and abortions. Previous attempts atcontrolling BHV-1 infection have utilized vaccines comprising liveattenuated virus (Gerber, J. D., et al., 1978, Am. J. Vet. Res.39:753-760; Mitchell, D., 1974, Can. Vet. Jour. 15:148-151), inactivatedvirus (Frerichs, G. N., et al., 1982, Vet. Rec. 111:116-122), and viralsubunits such as, e.g., one of the three major BHV-1 glycoproteins,which have been designated in the art as gI, gIII, and gIV (Babiuk, L.A., et al., 1987, Virology 159:57-66; van Drunen, S., et al., 1993,Vaccine 11:25-35). In addition, the ability of a recombinant, truncatedversion of the BHV-1 gIV glycoprotein (designated in the art as BHV-1tgIV) to induce mucosal immunity against BHV-1 has been demonstrated(van Drunen, S., et al., 1994, Vaccine, 12:1295-1302). However, theart-recognized BHV-1 vaccines are contraindicated for use in pregnantcattle, seropositive or seronegative, and also contraindicated for usein calves nursing pregnant cows.

[0004] BVDV Types 1 and 2 have been implicated in a variety of clinicalsyndromes. Studies have established that the virus causes severe primaryrespiratory disease; that persistently infected (PI) cattle are a majorsource of infection for susceptible calves; and that BVDV infects whitecell reservoirs, causing profound and broad-based deficits in the immunesystem. Ellis et al. (1996) JAVMA 208:393-400; Baum et al. (1993) TheCompendium Collection: Infectious Disease in Food Animal Practice.Trenton, N.J. Veterinary Learning Systems-1 13-121; Meyling et al.(1987) Agric Pestivirus Infect Rumin 225-231. Abortion or mummificationcan result when pregnant cattle become infected especially during thefirst trimester. Bolin et al. (1989) Am J. Vet Res 52:1033-1037. Mucosaldisease, another often fatal manifestation of bovine viral diarrhea(BVD), results from early fetal infection with a noncytopathic BVDVbiotype, development of immunotolerance to the virus, birth of apersistently infected (PI) calf, and subsequent superinfection with acytopathic BVDV biotype. Bolin et al. (1989) Am J. Vet Res 52:1033-1037.BVDV Type 2, once recognized chiefly as a hemorrhagic BVDV isolatemostly in dairy herds, has become the predominant strain isolated inmost regions of the United States from both BVD-related abortions andrespiratory cases. Van Oirschot et al. (1999) Vet Micro 64:169-183.

[0005] BVDV is classified in the pestivirus genus and Flaviviridaefamily. It is closely related to viruses causing border disease in sheepand classical swine fever. Infected cattle exhibit “mucosal disease”which is characterized by elevated temperature, diarrhea, coughing andulcerations of the alimentary mucosa (Olafson, et al., Cornell Vet.36:205-213 (1946); Ramsey, et al., North Am. Vet. 34:629-633 (1953)).The BVD virus is capable of crossing the placenta of pregnant cattle andmay result in the birth of PI calves (Malmquist, J. Am. Vet. Med. Assoc.152:763-768 (1968); Ross, et al., J. Am. Vet. Med. Assoc. 188:618-619(1986)). These calves are immunotolerant to the virus and persistentlyviremic for the rest of their lives. They provide a source for outbreaksof mucosal disease (Liess, et al., Dtsch. Tieraerztl. Wschr. 81:481-487(1974) and are highly predisposed to infection with microorganismscausing diseases such as pneumonia or enteric disease (Barber, et al.,Vet. Rec. 117:459-464 (1985).

[0006] According to BVDV virus growth studies in cultured cells, twoviral biotypes have been classified: viruses that induce a cytopathiceffect (cp) and viruses that do not induce a cytopathic effect (ncp) ininfected cells (Lee et al., Am. J. Vet. Res. 18: 952-953; Gillespie etal., Cornell Vet. 50: 73-79, 1960). Cp variants can arise from the PIanimals preinfected with ncp viruses (Howard et al., Vet. Microbiol. 13:361-369, 1987; Corapi et al., J. Virol. 62: 2823-2827, 1988). Based onthe genetic diversity of the 5′ non-translated-region (NTR) and theantigenic differences in the virion surface glycoprotein E2 of BVDviruses, two major genotypes have been proposed: type I and II. BVDVtype 1 represents classical or traditional virus strains which usuallyproduce only mild diarrhea in immunocompetent animals, whereas BVDV type2 are emerging viruses with high virulence which can producethrombocytopenia, hemorrhages and acute fatal disease (Corapi et al., J.Virol. 63: 3934-3943; Bolin et al., Am. J. Vet. Res. 53: 2157-2163;Pellerin et al., Virology 203: 260-268, 1994; Ridpath et al., Virology205: 66-74, 1994; Carman et al., J. Vet. Diagn. Invest. 10: 27-35,1998). Type I and II BVDV viruses have distinct antigenicity determinedby a panel of monoclonal antibodies (Mabs)and by cross-neutralizationusing virus-specific antisera raised in animals (Corapi et al., Am. J.Vet. Res. 51: 1388-1394, 1990). Viruses of either genotype may exist asone of the two biotypes, cp or ncp virus.

[0007] Studies from BVD virus infected animals suggest that BVD virusesinduce both B-cell and T-cell responses in animals (Donis et al.,Virology 158: 168-173, 1987; Larsson et al., Vet. Microbiol. 31:317-325, 1992; Howard et al., Vet. Immunol. Immunopathol. 32: 303-314,1992; Lambot et al., J. Gen. Virol. 78: 1041-1047, 1997; Beer et al.,Vet. Microbiology. 58: 9-22, 1997).

[0008] A number of BVDV vaccines have been developed using chemicallyinactivated BVD viral isolates (Fernelius et al., Am. J. Vet. Res. 33:1421-1431, 1972; Kolar et al., Am. J. Vet. Res. 33: 1415-1420, 1972;McClurkin et al., Arch. Virol. 58: 119, 1978). Multiple doses arerequired for the inactivated viral vaccines to achieve primaryimmunization. Some inactivated BVDV vaccines provide protection againstinfection by type I BVDV only (Beer et al., Vet. Microbiology.77:195-208, 2000). Fetal protection has not been achieved withinactivated BVDV vaccines due to a short duration of immunity and aninefficient cross-type protection (Bolin, Vet. Clin. North Am. FoodAnim. Pract. 11: 615-625,1995).

[0009] Modified-live virus (MLV) vaccines, on the other hand, offer ahigher level of protection. Currently, licensed BVDV MLV vaccines areproduced using attenuated viruses obtained via repeated passage inbovine or porcine cells (Coggins et al., Cornell Vet. 51: 539-, 1961;Phillips et al., Am. J. Vet. Res. 36: 135-, 1975), or using chemicallymodifiedviruses which exhibit a temperature-sensitive phenotype (Lobmannet al., Am. J. Vet. Res. 45: 2498-, 1984; 47: 557-561, 1986). A singledose of MLV vaccine is sufficient for immunization, and duration of theimmunity can last for years in vaccinated cattle. However, as thesevaccines have been developed using type I BVDV virus strains, theprotection is against type I virus only. Moreover, the available BVDVvaccines are not indicated for use in pregnant cattle or calves nursingpregnant cows.

[0010] PI₃ virus typically produces only mild disease when acting alone;however, the virus predisposes the respiratory tract to secondaryinfection with more pathogenic organisms including IBR virus, BRSV, andBVDV, resulting in the classic shipping fever syndrome. Of the variousviruses known to cause respiratory disease in cattle, PI₃ virus is themost widespread. Ellis et al. (1996) JAVMA 208:393-400.

[0011] BRSV has a preference for the lower respiratory tract, andseverity of infection is determined chiefly by the immune system'sresponse to key viral proteins. Bolin et al. (1990) Am J Vet Res 51:703.Affected cattle generally show nonspecific signs including serous nasaland ocular discharge, a mild, often biphasic fever, and dry, hackingcough. More severely affected cattle develop a harsh cough, showlabored, open-mouth breathing, and frothy saliva around the mouth, andmay quit eating and drinking. Ellis et al. (1996) JAVMA 208:393-400.

[0012] Leptospirosis, caused by spirochetes of the genus Leptospira, isan economically important zoonotic infection of livestock. Leptospiraborgpetersenii serovar hardjo (L. hardjo) and L. interrogans serovarpomona (L. pomona) are the two serovars most commonly associated withcattle leptosporosis worldwide. In one survey of US cattle, 29% reactedserologically with L. hardjo, and 23% with L. pomona. Leptospires invadethe body via mucous membranes or broken skin, and are disseminated viathe blood. They display tropisms for the kidney and genital tract, andless commonly the vitreous humor of the eye and the central nervoussystem. The most common means of infection is by direct or indirectcontact with infected urine, milk, or placental fluids, but venereal andtrans-ovarian transmission are also known. Leptospiral infection ofcattle may result in acute fever, agalactia, abortion, or birth ofpremature and weak infected calves, and may contribute to breedingfailures and low conception rates. Infections can be treated withantibiotics, but they may be inapparent in cattle that are not lactatingor pregnant. In such cattle they establish acute or chronic infection ofthe kidneys, resulting in urinary shedding of virulent organisms whichin turn may infect other animals or their human handlers. Immunity toLeptospira is serovar specific, and although vaccines have beenavailable for many years, most induce only a poor and short-livedimmunity.

[0013] There is therefore a need for development of combination vaccinesthat provide protection against a large variety of antigens that aresafe for pregnant and nursing cows and their offspring and meet dairyand beef cow market needs. The present invention provides vaccines forthe treatment and prevention of the major infectious causes ofrespiratory and reproductive disease in animals, such as cows andcalves. The present invention further provides immunogenic compositionsand methods of treating or preventing diseases or disorders in animals.

SUMMARY OF THE INVENTION

[0014] The present invention provides a method of treating or preventinga disease or disorder in an animal caused by infection with at least oneof, BVDV Type 1 or Type 2, BHV-1, PI3, BRSV, Campylobacter fetus,Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardjo-prajitno, Leptospira icterohaemmorrhagiae, Leptospiraborgpetersenii hardjo-bovis and Leptospira interrogans pomona comprisingadministering to the animal, an effective amount of a combinationvaccine.

[0015] The present method provides protection to animals such as bovine,in particular, dairy cattle, against respiratory infection andreproductive disease. The present method provides protection to animalssuch as pregnant cows against abortion caused by IBR and persistentfetal infections caused by BVDV, Types 1 and 2. The present method alsoprovides protection to animals such as lactating cows and calves nursingpregnant cows against persistent infections caused by BVDV, Types 1 and2. Thus, the present method provides protection to breeding age animals,pregnant animals and lactating animals.

[0016] The combination vaccine employed in the present methods can be awhole or partial cell preparation (e.g., modified live preparation). Thecombination vaccine administered in accordance with the presentinvention may include additional components, such as an adjuvant andoptionally a second or more antigens. A second antigen is selected fromthe following, including, but not limited, to bovine herpesvirus type 1(BHV-1), bovine viral diarrhea virus (BVDV type 1 or 2), bovinerespiratory syncitial virus (BRSV), parainfluenza virus (PI3),Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interroganspomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava,Campylobacter fetus, Neospora caninum, Trichomonus fetus, Mycoplasmabovis, Haemophilus somnus, Mannheimia haemolytica and Pasturellamultocida.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method of treating or preventinga disease or disorder in an animal caused by infection with IBR, BVDV,PI3, BRSV, Campylobacter fetus and/or Leptospirae by administering tothe animal, an effective amount of a combination vaccine.

[0018] In certain embodiments, the vaccines used in the method of thepresent invention comprise a modified live vaccine and apharmaceutically acceptable carrier, or a modified live vaccine and anadjuvant.

[0019] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections which describe or illustrate certain features, embodimentsor applications of the invention.

Definitions and Abbreviations

[0020] The term “treating or preventing” with respect to a disease ordisorder as used herein means reducing or eliminating the risk ofinfection by a virulent BVDV virus, types I and 2; IBR; P13; BRSV;Campylobacteria; and/or Leptospira antigens, ameliorating or alleviatingthe symptoms of an infection, or accelerating the recovery from aninfection. The treatment is considered therapeutic if there is areduction in viral or bacterial load, decrease in pulmonary infections,reduced rectal temperatures, and/or increase in food uptake and/orgrowth. The treatment is also considered therapeutic if there is areduction in fetal infection and urinary shedding due to infection withLeptospira serovars hardjo and pomona, for example.

[0021] The method of the present invention is, for example, effective inpreventing or reducing abortion caused by IBR and infections caused byBVDV Types 1 and 2, and reducing rectal temperatures. The presentinvention is therefore contemplated to provide fetal protection againstIBR and infections caused by BVDV Types 1 and 2 as well as fetalprotection against cattle herpes and cattle pestiviruses. The presentinvention is also contemplated to provide protection against persistentfetal infection, such as persistent BVDV infection. By “persistent fetalinfection” is meant infection occurring during early fetal development(e.g., 45-125 days of gestation) that leads to the live birth of animalsthat are immunotolerant to BVDV and maintain active BVDV replication andmultiplication that often occurs at a high rate for months or years,serving as a permanent source of BVDV in the herd. These persistentlyinfected animals are also at risk of developing fatal mucosal disease ifsuperinfected with a cytopathic virus biotype.

[0022] The term “combination vaccine” is meant a bivalent or multivalentcombination of antigens including modified live antigens and/orinactivated antigens. In accordance with the present invention acombination vaccine can comprise modified live infectious IBR, PI3, BRSVand inactivated BVDV Types 1 and 2, one or more antigens such as but notlimited to Leptospira canicola, Leptospira grippotyphosa, Leptospiraborgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia,Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis,Leptospira bratislava, Campylobacter fetus, Neospora caninum,Trichomonus fetus, Mycoplasma bovis, Haemophilus somnus, Mannheimiahaemolytica and Pasturella multocida, a veterinary acceptable carrierand an adjuvant. In a preferred embodiment the modified live IBRcomponent is temperature sensitive IBR. In another preferred embodimentthe BVDV Type 2 component is cytopathic (cpBVD-2 strain 53637-ATCC No.PTA-4859) and the BVDV Type 1 component is cytopathic 5960 (cpBDV-1strain 5960-National Animal Disease Center, United States Department ofAgriculture, Ames, Iowa). The present invention also contemplatesnon-cytopathic BVDV Type 1 and Type 2 strains. In still anotherpreferred embodiment, the modified live antigens are desiccated,lyophilized or vitrified.

[0023] In accordance with the present invention a combination vaccinecan comprise inactivated BVDV Types 1 and 2, one or more antigens suchas, but not limited to, Leptospira canicola, Leptospira grippotyphosa,Leptospira borgpetersenii hardio-prajitno, Leptospiraicterohaemmorrhagia, Leptospira interrogans pomona, Leptospiraborgpetersenii hardjo-bovis, Leptospira bratislava, Campylobacter fetus,Neospora caninum, Trichomonus fetus, Mycoplasma bovis, Haemophilussomnus, Mannheimia haemolytica and Pasturella multocida, a veterinaryacceptable carrier and an adjuvant. The term “combination vaccine” asused herein also refers to a multicomponent composition containing atleast one modified live antigen, at least one second antigen and anadjuvant which prevents or reduces the risk of infection and/or whichameliorates the symptoms of infection. In a preferred embodiment thesecond antigen is inactivated. In a preferred embodiment the source ofthe combination vaccine is PregSure® 5 (Pfizer, Inc.), PregSure® 5-L5(Pfizer, Inc.) and PregSure® 5-VL5 (Pfizer, Inc.). A particularlypreferred source of the combination vaccine is PregSure® 5-VL5.

[0024] The protective effects of a combination vaccine compositionagainst a pathogen are normally achieved by inducing in the subject animmune response, either a cell-mediated or a humoral immune response ora combination of both. Generally speaking, abolished or reducedincidences of BVDV, IBR, and/or PI3 infection, amelioration of thesymptoms, or accelerated elimination of the viruses from the infectedsubjects are indicative of the protective effects of a combinationvaccine composition. The vaccine compositions of the present inventionprovide protection against infections caused by either or both type 1and type 2 BVD viruses as well as abortions caused by BHV-1 (IBR) andrespiratory infections caused by PI3 and BRSV.

[0025] The present method of treating or preventing a disease ordisorder in an animal caused by infection with IBR, BVDV, PI3, BRSV,Campylobacter fetus and/or Leptospirae by administering a combinationvaccine is also referred to herein as a vaccination method.

[0026] The term “combination vaccine” that may be used in the presentmethod can include, for example, an inactivated whole or partial C.fetus and/or Leptospira cell preparation, inactivated BVDV types 1 and 2and/or one or more modified live antigens such as BHV-1, PI3 and/orBRSV.

[0027] In one embodiment, the vaccine compositions of the presentinvention include an effective amount of one or more of theabove-described BVDV viruses, preferably cpBVD-2 strain 53637 (ATCC No.PTA4859); cpBVD-1 strain 5960 (cpBDV-1 strain 5960-National AnimalDisease Center, United States Department of Agriculture, Ames, Iowa);IBRts mutant strain RBL 106 (National Institute of Veterinary Research,Brussels, Belgium); PI₃ ts mutant strain RBL 103 (RIT, Rixensart,Belgium); BRSV strain 375 (Veterinary Medical Research Institute, Ames,Iowa) Purified BVDV viruses can be used directly in a vaccinecomposition, or preferably, BVD viruses can be further attenuated by wayof chemical inactivation or serial passages in vitro. Typically, avaccine contains between about 1×10³ and about 1×10¹⁰ plaque or colonyforming units of virus, with a veterinary acceptable carrier and anadjuvant, in a volume of between 0.5 and 5 ml and preferably about 2 ml.The precise amount of a virus in a vaccine composition effective toprovide a protective effect can be determined by a skilled veterinaryphysician. Veterinary acceptable carriers suitable for use in vaccinecompositions can be any of those described hereinbelow.

[0028] The typical route of administration will be intramuscular orsubcutaneous injection of between about 0.1 and about 5 ml of vaccine.The vaccine compositions of the present invention can also includeadditional active ingredients such as other vaccine compositions againstBVDV, e.g., those described in WO 9512682, WO 9955366, U.S. Pat. No.6,060,457, U.S. Pat. No. 6,015,795, U.S. Pat. No. 6,001,613, and U.S.Pat. No. 5,593,873.

[0029] Vaccination can be accomplished by a single inoculation orthrough multiple inoculations. If desired, sera can be collected fromthe inoculated animals and tested for the presence of antibodies to BVDvirus.

[0030] In another embodiment of the present invention, the vaccinecompositions are used in treating BVDV infections. Accordingly, thepresent invention provides methods of treating infections in animalsubjects caused by BVD viruses of type 1 or type 2, or a combination oftype 1 and type 2, by administering to an animal, a therapeuticallyeffective amount of a BVD virus of the present invention. In anotherembodiment the vaccine compositions of the present invention areeffective for the improvement of herd fertility, and for the reductionof the risk of disease transmission from cattle to human handlers.

[0031] By “animal subject” is meant to include any animal that issusceptible to BVDV, BHV, PI₃, BRSV or Leptospira infections, forexample, such as bovine, sheep and swine.

[0032] In practicing the present methods, a vaccine composition of thepresent invention is administered to a cattle preferably viaintramuscular or subcutaneous routes, although other routes ofadministration can be used as well, such as e.g., by oral, intranasal(e.g. aerosol or other needleless administration), intra-lymph node,intradermal, intraperitoneal, rectal or vaginal administration, or by acombination of routes. Boosting regimens may be required and the dosageregimen can be adjusted to provide optimal immunization.

[0033] By “immunogenic” is meant the capacity of a BVD virus to provokean immune response in an animal against type 1 or type 2 BVD viruses, oragainst both type 1 and type 2 BVD viruses. The immune response can be acellular immune response mediated primarily by cytotoxic T-cells, or ahumoral immune response mediated primarily by helper T-cells, which inturn activates B-cells leading to antibody production.

[0034] According to the present invention, the viruses are preferablyattenuated by chemical inactivation or by serial passages in cellculture prior to use in an immunogenic composition. The methods ofattenuation are well known to those skilled in the art.

[0035] A preferred virus to be included in an immunogenic composition ofthe present invention is BVDV cp53637(ATCC No. PTA-4859). Anotherpreferred virus to be included in an immunogenic composition of thepresent invention is BVDV 5960. A further preferred virus to be includedin an immunogenic composition of the present invention is IBR strain tsmutant strain RBL 106. Another preferred virus to be included in animmunogenic composition of the present inventions is PI₃ ts mutantstrain RBL 103. Yet another preferred virus to be included in animmunogenic composition of the present invention is BRSV strain 375.

[0036] The immunogenic compositions of the present invention can alsoinclude additional active ingredients such as other immunogeniccompositions against BVDV, e.g., those described in copendingapplication Ser. No. 08/107,908, WO 9512682, WO 9955366, U.S. Pat. No.6,060,457, U.S. Pat. No. 6,015,795, U.S. Pat. No. 6,001,613, and U.S.Pat. No. 5,593,873.

[0037] In addition, the immunogenic and vaccine compositions of thepresent invention can include one or more veterinary-acceptablecarriers. As used herein, “a veterinary-acceptable carrier” includes anyand all solvents, dispersion media, coatings, adjuvants, stabilizingagents, diluents, preservatives, antibacterial and antifungal agents,isotonic agents, adsorption delaying agents, and the like. Diluents caninclude water, saline, dextrose, ethanol, glycerol, and the like.Isotonic agents can include sodium chloride, dextrose, mannitol,sorbitol, and lactose, among others. Stabilizers include albumin, amongothers. Adjuvants include, but are not limited to, the RIBI adjuvantsystem (Ribi Inc.), alum, aluminum hydroxide gel, Cholesterol, oil-inwater emulsions, water-in-oil emulsions such as, e.g., Freund's completeand incomplete adjuvants, Block co-polymer (CytRx, Atlanta Ga.), SAF-M(Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.) or other saponin fractions,monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labileenterotoxin from E. coli (recombinant or otherwise), cholera toxin, ormuramyl dipeptide, among many others. The immunogenic compositions canfurther include one or more other immunomodulatory agents such as, e.g.,interleukins, interferons, or other cytokines. The immunogeniccompositions can also include Gentamicin and Merthiolate. While theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan, the present invention contemplates compositionscomprising from about 50 μg to about 2000 μg of adjuvant and preferablyabout 500 μg/2 ml dose of the vaccine composition. In another preferredembodiment, the present invention contemplates vaccine compositionscomprising from about 1 pg/ml to about 60 μg/ml of antibiotic, and morepreferably less than about 30 μg/ml of antibiotic.

[0038] The immunogenic compositions of the present invention can be madein various forms depending upon the route of administration. Forexample, the immunogenic compositions can be made in the form of sterileaqueous solutions or dispersions suitable for injectable use, or made inlyophilized forms using freeze-drying techniques. Lyophilizedimmunogenic compositions are typically maintained at about 4° C., andcan be reconstituted in a stabilizing solution, e.g., saline or andHEPES, with or without adjuvant.

[0039] The immunogenic compositions of the present invention can beadministered to animal subjects to induce an immune response againsttype 1 or type 2 BVD viruses, or against both type 1 and type 2 BVDviruses. Accordingly, another embodiment of the present inventionprovides methods of stimulating an immune response against type 2 ortype 2 BVD viruses, or against a combination of type 1 and type 2 BVDviruses by administering to an animal subject an effective amount of animmunogenic composition of the present invention described above. By“animal subject” is meant to include any animal that is susceptible toBVDV infections, such as bovine, sheep and swine.

[0040] In accordance with the methods of the present invention, apreferred immunogenic composition for administration to an animalsubject includes the BVDV cp53637 virus and/or the BVDV cp5960 virus. Animmunogenic composition containing a BVDV virus, preferably attenuatedby chemical inactivation or serial passage in culture, is administeredto a cattle preferably via intramuscular or subcutaneous routes,although other routes of administration can be used as well, such ase.g., by oral, intranasal, intra-lymph node, intradermal,intraperitoneal, rectal or vaginal administration, or by a combinationof routes.

[0041] Immunization protocols can be optimized using procedures wellknown in the art. A single dose can be administered to animals, or,alternatively, two or more inoculations can take place with intervals oftwo to ten weeks. Depending on the age of the animal, the immunogenic orvaccine composition can be readministered. For example, the presentinvention contemplates the vaccination of healthy cattle prior to sixmonths of age and revaccination at six months of age. In anotherexample, the present invention contemplates the vaccination ofprebreeding cattle at about 5 weeks prebreeding and again at about 2weeks prebreeding to protect a fetus against infection caused by BVDVTypes 1 and 2. Semiannual revaccination with a single dose of thecombination vaccine is also contemplated to prevent BVDV fetalinfection.

[0042] The extent and nature of the immune responses induced in thecattle can be assessed by using a variety of techniques. For example,sera can be collected from the inoculated animals and tested for thepresence of antibodies specific for BVDV viruses, e.g., in aconventional virus neutralization assay.

[0043] The term “cattle” as used herein refers to bovine animalsincluding but not limited to steer, bulls, cows, and calves. Cattle asused herein refers to pregnant and lactating bovine animals. Preferably,the method of the present invention is applied to an animal which is anon-human mammal; preferably, a lactating or pregnant cow and its fetus.

[0044] The term “therapeutically effective amount” or “effective amount”refers to an amount of combination vaccine sufficient to elicit animmune response in the animal to which it is administered. The immuneresponse may comprise, without limitation, induction of cellular and/orhumoral immunity. The amount of a vaccine that is therapeuticallyeffective may vary depending on the particular virus used, the conditionof the cattle and/or the degree of infection, and can be determined by aveterinary physician.

Inactivated (Partial or Whole Cell) and Modified Live Vaccines

[0045] Inactivated or modified live vaccines for use in the method ofthe present invention can be prepared using a variety of methods whichare known in the art.

[0046] For example, BVDV isolates can be obtained directly from infectedcow uteri using known techniques.

[0047] BVDV isolates can be inactivated using a variety of knownmethods, e.g., treating the bacterial isolate with binary ethyleneimine(BEI) as described in U.S. Pat. No. 5,565,205, or inactivation withformalin, glutaraldehyde, heat, irradiation, BPL, or other inactivatingagents known to the art.

[0048] In addition to inactivated viral isolates, a vaccine product canalso include an appropriate amount of one or more commonly usedadjuvants. Suitable adjuvants may include, but are not limited to:mineral gels, e.g., aluminum hydroxide; surface active substances suchas lysolecithin; glycosides, e.g., saponin derivatives such as Quil A orGPI-0100; pluronic polyols; polyanions; non-ionic block polymers, e.g.,Pluronic F-127 (B.A.S.F., USA); peptides; mineral oils, e.g. MontanideISA-50 (Seppic, Paris, France), carbopol, Amphigen, Amphigen Mark II(Hydronics, USA), Alhydrogel, oil emulsions, e.g. an emulsion of mineraloil such as BayolF/Arlacel A and water, or an emulsion of vegetable oil,water and an emulsifier such as lecithin; alum; bovine cytokines;cholesterol; and combinations of adjuvants. In a preferred embodiment,the saponin containing oil-in-water emulsion is conventionallymicrofluidized.

[0049] A particularly preferred source of BVDV type 1, for use in thevaccine and method of the present invention is PregSure® (PFIZER INC.),containing BVDV strain 5960 (acquired from the National Animal DiseaseCenter (NADC), USDA, Ames, Iowa). A particularly preferred source ofBVDV type 2, for use in the vaccine and method of the present inventionis PregSure® (Pfizer, Inc.), containing BVDV strain 53637 (ATCC No.PTA-4859), acquired from the University of Guelph, Guelph, Ontario.

[0050] Preferably, the strains 5960 and 53637 are inactivated with BEIand adjuvanted with a commercially available adjuvant, preferably, QuilA-Cholesterol-Amphigen (Hydronics, USA). A preferred dose of theimmunogenic and vaccine compositions of the present invention is about2.0 ml. Preservatives can be included in the methods and compositions ofthe present invention. Preservatives contemplated by the presentinvention include gentamicin and merthiolate. A carrier can also beadded, preferably, PBS. Preparation of modified live vaccines, such asby attenuation of virulent strains by passage in culture, is known inthe art.

[0051] Inactivated BVDV isolates can also be combined with the followingbacteria and viruses, including but not limited to, bovine herpesvirustype 1 (BHV-1), bovine respiratory syncitial virus (BRSV), parainfluenzavirus (PI3), Campylobacter fetus, Leptospira canicola, Leptospiragrippotyphosa, Leptospira borgpetersenii hardjo-prajitno, Leptospiraicterohaemmorrhagiae, Leptospira borgpetersenii hardjo-bovis andLeptospira interrogans pomona.

Dosing and Modes of Administration

[0052] According to the present invention, an effective amount of acombination vaccine administered to cattle, including pregnant cows andcalves nursing pregnant cows provides effective immunity against diseaseand fetal infection associated with Bovine Viral Diarrhea Virus (Type 1and 2). In one embodiment, the combination vaccine is administered tocalves in two doses at an interval of about 3 to 4 weeks. For example,the first administration is performed when the animal is about 1 toabout 3 months of age. The second administration is performed about 1 toabout 4 weeks after the first administration of the combination vaccine.

[0053] In a preferred embodiment, the first administration is performedabout 5 weeks prior to animal breeding. The second administration isperformed about 2 weeks prior to animal breeding. Administration ofsubsequent vaccine doses is preferably done on an annual basis. Inanother preferred embodiment, animals vaccinated before the age of about6 months should be revaccinated after 6 months of age. Administration ofsubsequent vaccine doses is preferably done on an annual basis.

[0054] The amount of combination vaccine that is effective depends onthe ingredients of the vaccine and the schedule of administration.Typically, when an inactivated Bovine Viral Diarrhea Virus preparationis used in a vaccine, an amount of the vaccine containing about 10³ toabout 10¹⁰ colony forming units per dose of BVDV, and preferably about10⁵ to about 10⁸ colony forming units per dose of BVD V(Type 1 and 2) iseffective when administered twice to the animal during a period of about3 to 4 weeks. Preferably, a combination vaccine that provides effectiveimmunity contains about 10⁵ to 10⁸ colony forming units/dose of BVDV(Type 1 and 2) and more preferably, about 10⁶ colony forming units/dose,when administered twice to the animal during a period of about 3 to 4weeks. The first administration is performed about 5 weeks prior toanimal breeding. The second administration is performed about 2 weeksprior to animal breeding. Administration of subsequent vaccine doses ispreferably done on an annual basis. Animals vaccinated before the age ofabout 6 months should be revaccinated after 6 months of age.Administration of subsequent vaccine doses is preferably done on anannual basis.

[0055] According to the present invention, when the preferred product,PregSure® 5 (Pfizer, Inc.), is administered, PregSure® 5 is administeredpreferably twice, each time in the amount of about 0.5 ml to about 5.0ml, preferably about 1.5 ml to about 2.5 ml, and more preferably, about2 ml. When the preferred product PregSure® 5-L5 or PregSure® 5-VL5 isadministered, PregSure® 5-L5 or PregSure® 5-VL5 is administeredpreferably twice, each time in the amount of about 0.5 ml to about 10.0ml, preferably about 3 ml to about 7 ml, and more preferably, about 5ml. The first administration is performed about 5 weeks prior to animalbreeding. The second administration is performed about 2 weeks prior toanimal breeding. Administration of subsequent vaccine doses ispreferably done on an annual basis. Animals vaccinated before the age ofabout 6 months should be revaccinated after 6 months of age.Administration of subsequent vaccine doses is preferably done on anannual basis.

[0056] In accordance with the present invention, administration can beachieved by known routes, including the oral, intranasal, topical,transdermal, and parenteral (e.g., intravenous, intraperitoneal,intradermal, subcutaneous or intramuscular). A preferred route ofadministration is subcutaneous or intramuscular administration.

[0057] The present invention also contemplates a single primary dosefollowed by annual revaccination, which eliminates the necessity ofadministration of additional doses to calves prior to annualrevaccination in order to generate and/or maintain immunity againstinfection.

[0058] The combination vaccine administered in accordance with thepresent invention can include additional components, such as an adjuvant(e.g., mineral gels, e.g., aluminum hydroxide; surface active substancessuch as Cholesterol, lysolecithin; glycosides, e.g., saponin derivativessuch as Quil A, QS-21 or GPI-0100; pluronic polyols; polyanions;non-ionic block polymers, e.g., Pluronic F-127; peptides; mineral oils,e.g. Montanide ISA-50, carbopol, Amphigen, Alhydrogel, oil emulsions,e.g. an emulsion of mineral oil such as BayolF/Arlacel A and water, oran emulsion of vegetable oil, water and an emulsifier such as lecithin;alum; bovine cytokines; and combinations of adjuvants.).

[0059] According to the present invention, the administration of aneffective amount of a combination vaccine administered to cattle atapproximately 3 months of age provides effective immunity againstrespiratory infections and reproductive disease, and reduces abortions.The present invention also provides a method of immunizing cattle,including but not limited to cows, calves, and prebreeding heifers,against infection caused by BVDV (types 1 and 2), and respiratorydisease attributed to IBR, BVDV (Types 1 and 2), P13, BRSV,campylobacteriosis and leptospiriosis comprising administering to theanimal at least one dose, and preferably two doses of the combinationvaccine in order to immunize the animal against infection caused by BVD(types 1 and 2), IBR, PI3, BRSV, Leptospira canicola, Leptospiragrippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospiraicterohaemmorrhagia, Leptospira interrogans pomona, Leptospiraborgpetersenii hardjo-bovis, Leptospira bratislava, and Campylobacterfetus.

[0060] In a preferred embodiment, the vaccine is administeredsubcutaneously. In another preferred embodiment, the vaccine isadministered intramuscularly. Moreover, it is preferred that the vaccinedose comprise about 2 ml to about 7 ml, and preferably about 5 ml , eachml containing about 10³ to about 10¹⁰ colony forming units/per dose ofvirus. In another preferred embodiment the vaccine comprises about 2 ml,each ml containing about 10³ to about 10¹⁰ colony forming units per doseof virus. The combination vaccine is desirably administered twice to theanimal; once at about 1 to about 3 months of age, and once at about 1 to4 weeks later. The present invention also contemplates semiannualrevaccinations with a single dose and revaccination prior to breeding.

[0061] The present invention also provides a method of protecting bovinefetuses against fetal infection and persistent fetal infection,comprising administering to the animal at least one dose, and preferablytwo doses of the combination vaccine in order to immunize the fetusagainst infection caused by BVD (types 1 and 2), IBR, PI3, BRSV,Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interroganspomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava,and Campylobacter fetus. The combination vaccine is desirablyadministered twice to the animal, once about five weeks prior tobreeding and once at about two weeks prior to breeding.

[0062] The present invention also contemplates that the administrationof an effective amount of a combination vaccine administered to animals,and preferably cattle to treat or prevent disorders including persistentfetal infections and reproductive disorders, such as abortions in suchanimals.

[0063] The present invention is further illustrated, but not limited bythe following examples.

EXAMPLE 1 Materials and Methods

[0064] Animals—Fifty-six BVDV seronegative (i.e., having serumneutralization [SN] titers <1:2) cows suitable for breeding wereobtained from multiple sources and maintained in research isolationfacilities for the duration of the study. Each animal was identifiedwith duplicate ear tags, one placed in each ear. New tags were installedin cases where an animal lost an ear tag. Prior to the study, testanimals were inoculated with commercial vaccines for leptospirosis,campylobacteriosis (vibriosis), and clostridial infections. Test animalswere maintained under supervision of an attending veterinarian, whoclinically monitored them on a daily basis.

[0065] Test vaccine—The test vaccine was a multivalent, modified liveinfectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) vaccine in desiccated form,rehydrated with an inactivated, liquid BVDV vaccine combined with anadjuvant. (Pfizer Inc, New York, N.Y.) The BVDV component contained theminimum BVDV-1 and -2 immunizing doses, combined with a sterileadjuvant. Potency of the BVDV immunizing antigens was established bycalculating the geometric mean titer (GMT) for 8 replicate titrations ofthe bulk fluid used for vaccine preparation. Following rehydration, theIBR-PI3-BRSV-BVDV vaccine was administered in 2 mL doses by eitherintramuscular (IM) or subcutaneous (SC) injection. The desiccatedIBR-PI3-RSV vaccine reconstituted with sterile water was used as aplacebo, and was given by IM injection.

[0066] Challenge virus—A noncytopathic BVDV type 2 field isolate (Strain94B-5359, obtained from Dr. Hana Van Campen, Wyoming State VeterinaryLaboratory, University of Wyoming) was used as a challenge agent. Virusidentity was confirmed by SN assay and reverse transcriptase polymerasechain reaction (RT-PCR). The RT-PCR analysis was positive for BVDV type2 nucleotide sequences for the p125 protein and the 5 untranslatedregion, and negative for the BVDV type 1 gp53 and p80 conservedsequences. Challenge virus potency was established at a GMT of 10^(3.2)TCID₅₀/mL by 2 replicate titrations made immediately before and afterchallenge. Challenge inoculum was given intranasally in a 4 mL divideddose, 2 mL per nostril.

[0067] Serologic assays—Serum neutralization titers for BVDV types 1 and2 were determined by a constant-virus, decreasing-serum assay in bovinecell culture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 125c.

[0068] Virus isolation—Postchallenge (PC) isolation of BVDV in bovinecell culture was attempted from peripheral cow blood, amniotic fluid,fetal blood, and fetal tissues. A BVDV-positive cell culture wasdetermined by indirect immunofluorescence using goat anti-BVDVpolyclonal antibodies. Isolation of BVDV from fetal tissues was alsoattempted using immunohistochemistry methods previously described.(Haines D M, Clark E G, and Dubovi E J. Vet Pathol 1992;29:27-32.) Wholeblood from cows was drawn from the jugular vein in 5-10 mL samples andplaced in heparin-containing tubes for preparation of buffy coat cellsused for virus isolation attempts. Amniotic fluid was collected underlocal anesthesia by left-flank laparotomy and aspiration of a 3-5 mLsample from the uterus.

[0069] Following caesarian section or spontaneous abortion, the eyes,spleen, thymus, and 3 brain sections (brainstem/midbrain, cerebrum, andcerebellum) were aseptically collected from each fetus. Supernatant fromhomogenized fetal tissues was used for virus isolation attempts in cellculture. For purposes of immunohistochemistry evaluation, fetal tissueswere embedded in paraffin and tested in duplicate using 1:800 and 1:600ascites dilutions containing anti-BVDV monoclonal antibodies.

[0070] Biometric data analysis—To demonstrate protection followingchallenge, a statistically significant reduction in incidence ofmaternal and fetal BVDV type 2 infection had to be demonstrated invaccinated groups (T2 and T3) versus the placebo control group (T1). AFisher's exact test was used to compare incidence of (1) cow viremiaduring the first 14 days following challenge, (2) BVDV isolation fromamniotic fluid, (3) BVDV isolation from fetal tissue and fetal bloodfollowing spontaneous abortion or caesarian section, and (4)BVDV-positive fetal tissue immunohistochemistry. Serum neutralizationtiters were analyzed using a mixed linear model with repeated measures.Least squares means from the analysis of variance were used to calculatea geometric mean titer (GMT), which excluded SN data for cows that werenot challenged. A probability value of P≦0.05 was used to determinestatistical significance.

[0071] Fetal protection study—The 56 test cows were randomly assigned toone of three test groups, an IM placebo group (T1), an IM vaccinationgroup (T2), and a SC vaccination group (T3) as noted in Table 1. Cowswere inoculated with either vaccine or placebo on study Day 0 and Day21. In all cases, the Day 0 inoculation was administered on the leftside of the neck, and the Day 21 inoculation was administered on theright side of the neck.

[0072] On Day 1, cows were given feed top-dressed with melengestrolacetate for 14 days. On Day 32, all cows received an IM prostaglandininjection (Lutalyse, Pharmacia & Upjohn, Kalamazoo, Mich.) tosynchronize estrus. Cows which displayed estrus were bred by artificialinsemination with certified BVDV-negative semen. On Day 100, atapproximately 65 days of gestation, the pregnancy status of cows wasdetermined by rectal palpation. On Day 105, 23 cows with confirmedpregnancies were randomly selected from each test group (7 controls, 8IM vaccinates, and 8 SC vaccinates), relocated to a nearby isolationfacility, (Midwest Veterinary Services, Oakland, Nebr.) and commingled.On Day 119, the 23 test cows were challenged by intranasal inoculationof virulent BVDV. Blood samples were collected on the day of challengeand at 8 PC intervals, on Days 119, 121, 123, 125, 127, 129, 133, 140,and 147 (PC days 0, 2, 4, 6, 8, 10, 14, 21, and 28) for purposes of BVDVisolation and serologic assay.

[0073] On Day 147 (28 days after challenge), left flank laparotomieswere performed and amniotic fluid was extracted from each cow. On Day297, approximately 7-14 days prior to anticipated calving, test cowswere transported to facilities at the University of Nebraska, Departmentof Veterinary and Biomedical Services, for caesarian section.Immediately prior to surgery, a blood sample was collected from each cowfor SN assay. Following caesarian delivery (on Days 300-301), a bloodsample was collected from each fetus. Fetuses were then euthanized andtissues were aspecticaly collected for purposes of BVDV isolation.

[0074] In some cases where spontaneous abortions occurred, blood sampleswere taken from the dam when abortion was detected and two weeks later.The paired blood samples were submitted for serologic testing(University of Nebraska Veterinary Diagnositic Center, Lincoln, Nebr.)and aborted fetuses were evaluated for BVDV isolation (Pfizer CentralResearch, Lincoln, Nebr.) and histopathologic evaluation of fetaltissues (Saskatoon Veterinary Biodiagnostics, Saskatoon, SK, Canada).

Results

[0075] No adverse events were observed during or immediately followingadministration of the 2 vaccine doses.

[0076] All cows were seronegative to BVDV types 1 and 2 prior tovaccination (Day 0), confirming that the test animals wereimmunologically naive to BVDV at the outset of the study. The GMT valuesfor BVDV type 1 are shown in Table 2. Fifteen of 16 vaccinatesseroconverted following 2 vaccine doses. Cow number 61 (T3 group) hadBVDV type 1 SN titers of <1:2 on Day 0, 1:19 on Day 21, and <1:2 on Day33 and Day 119. The GMT values to BVDV type 2 are shown in Table 3. Allvaccinates, including cow 61, seroconverted following the second dose.(Cow 61 had BVDV type 2 SN titers of <2 on Day 0, 1:19 on Day 21,1:2,048 on Days 33, and 1:431 on Day 119.) At breeding (Days 32-41),vaccinated cows (excluding cow 61) had BVDV type 1 SN titers rangingfrom 1:64 to 1:13,777. Vaccinated cows had BVDV type 2 titers rangingfrom 1:64 to 1:6,889 at abreeding. Following vaccination, the differencein GMT values for the IM (T2) versus SC (T3) groups was notstatistically significant at either of 2 prechallenge intervals orfollowing challenge. All cows in the placebo group (T1) remainedseronegative for both BVDV types 1 and 2 up to the time of challenge(Day 119), indicating that the study was not compromised by adventitiousexposure. All placebo cows responded serologically to challenge,verifying that a viable challenge occurred in each animal. The placebogroup had a PC-GMT for BVDV type 1 that was significantly lower than theanemnestic responses achieved by either vaccine group (Table 2, Day147). Placebo cows also had a lower PC-GMT to BVDV type 2 compared toeither vaccine group, but the difference was statistically significantonly versus the IM (T2) vaccinates.

[0077] Twenty-three cows in the 3 test groups were confirmed pregnant,challenged, and subjected to amniocentesis (Table 1). Between the timeof amniocentesis (Day 147) and caesarian section (Day 300-301), 7 cowsaborted, 4 from the T2 group and 3 from the T3 group. In addition, 3bred cows (1 T1 placebo cow and 2 cows from the T3 group) were found tobe not pregnant at the time of caesarian section. These 3 cows wereconfirmed pregnant by rectal palpation on Day 100, approximately 65 daysafter breeding, indicating that subsequent undetected abortion or fetalresorption occurred. Because fetal tissues from the 3 cows with failedpregnancies were not available for evaluation, these animals wereremoved from the study. On Day 259, T1 placebo cow number 67 died, andits fetus was removed for purposes of BVDV isolation. Thus, at theconclusion of the study, 12 of the 23 cows that had been challengedunderwent caesarian section. The 12 caesarian derived fetuses plus the 7aborted fetuses and the fetus from the dead cow (20 in all) wereevaluated for BVDV isolation.

[0078] Cow 38 from the T2 group aborted its fetus on Day 156 (at 123days of gestation, and 37 days after challenge). Paired serum sampleswere not evaluated, but cow 38 was negative for postchallenge BVDVisolation from peripheral blood and amniotic fluid. The fetus wasseverely autolyzed. Histopathologic and bacteriologic evaluation of thefetus revealed purulent inflammation of the chorionic and subchorionicconnective tissues. Staphylococcus hyicus was isolated from the lung,liver, and thoracic fluid. Negative BVDV isolation andimmunohistochemistry results were obtained, indicating that the fetuswas not infected as a result of challenge.

[0079] Three T3 cows (numbers 21, 27, and 40) aborted on Days 158 or 159(at 125-127 days of gestation, and 39 or 40 days after challenge). Theabortions were not observed, so the recovered fetuses could not beattributed to specific cows. They were designated unknown fetuses 1, 2or 3. Unknown fetus 1 was mummified, with lesions histologically typicalof neosporosis. Unknown fetus 2 was autolyzed, with multifocal purulentand necrotizing placentitis and large numbers of bacterial cocci mixedwith inflammatory exudate. Staphylococcus hyicus was isolated from lung,kidney, liver, stomach contents and placental tissue. Unknown fetus 3was macerated and autolyzed. Staphylococcus sp was isolated from thelung, liver, kidney, and stomach contents. Negative BVDV isolation andimmunohistochemistry results were obtained for all tissues from the 3unknown fetuses. All 3 dams were negative for postchallenge BVDVisolation from peripheral blood. Cows 21 and 40 were likewise negativefor BVDV isolation from amniotic fluid, but cow 27 had a BVDV-positiveamniotic fluid sample. Paired serum samples from the dams were notevaluated.

[0080] Cow 45 from the T2 group aborted its fetus on Day 160 (at 128days of gestation, and 41 days after challenge). Extensive fetalautolysis was evident. Staphylococcus sp was isolated from the lung,liver, kidney, stomach contents, and placenta. Placentitis withmultifocal thrombosis and suppurative vascultisis was present. Serologicassays of thoracic fluid were negative for IBR, bovine viral diarrhea(BVD), and leptospirosis. Negative BVDV isolation andimmunohistochemistry results were obtained for all fetal tissues.

[0081] Cow 66 from the T2 group aborted its fetus on Day 195 (at 160days of gestation, and 76 days after challenge). Marked suppurativeinflammation of the placental lamina propria extending from the fetalsurface was observed. E. coli and Proteus vulgaris were cultured fromthe stomach contents and placenta. Paired serum samples and thoracicfluid serology results did not support IBR, BVD, or leptospirosis as anetiology. Negative BVDV isolation and immunohistochemistry results wereobtained for all fetal tissues.

[0082] Cow 31 from the T2 group aborted its fetus on Day 295 (at 262days of gestation, and 176 days after challenge). Histopathologicexamination revealed diffuse necropurulent placentitis, necrosis of thechorionic epithelium, and intense neutrophilic inflammation.Gram-negative coccobacilli and rods were cultured from the inflammatoryfoci. Paired serology results did not support IBR, BVD, or leptospirosisas an etiology. Negative BVDV isolation and immunohistochemistry resultswere obtained for all fetal tissues.

[0083] Positive BVDV isolation was obtained from PC peripheral bloodsamples and amniotic fluid obtained from T1 placebo cow 67, which diedprior to the conclusion of the study. All fetal tissues from this cowwere BVDV isolation and immunohistochemistry positive.

[0084] Blood samples collected from the 12 caesarian derived fetuseswere assayed for SN titers to BVDV type 1 and 2. None of the caesarianderived fetuses from the 5 placebo cows or 7 vaccinates was seropositivefor either BVDV type 1 or 2.

[0085] Postchallenge virus isolation results are shown in Table 4. All 7T1 placebo cows experienced BVDV viremia, corroborating serologicresults indicating that a viable challenge occurred in each of thenonvaccinated animals. Blood samples from all 16 of the T2 and T3vaccinates were negative for BVDV viremia on each of 8 postchallengesamples. The difference in the rate of PC viremia in T2 and T3vaccinates versus controls was statistically significantly (P≦0.0001).

[0086] Amniotic fluid from 2 of 16 (12.5 percent) vaccinates was BVDVpositive, versus positive results for 7 of 7 (100 percent) T1 placebocows, a statistically significant difference (P≦0.0001). Amniotic fluidsamples from T3 vaccinates 27 and 60 were positive. The fetus from cow27 was BVDV-negative by virus isolation and immunohistochemistrymethods.

[0087] Fetal tissues from 1 of 14 vaccinates (7.1 percent), T3 cow 60,were positive for BVDV isolation. This compared to BVDV isolation in 6of 6 (100 percent) fetuses from T1 placebo cows, a statisticallysignificant difference (P≦0.0001). BVDV isolation results were eitherall positive or all negative for the fetal tissues evaluated from eachfetus.

[0088] Immunohistochemistry results were BVDV positive for fetal tissuesfrom 1 of 14 (7.1 percent) T2 and T3 vaccinates (T3 cow 60). All fetaltissues from 6 of 6 (100 percent) T1 placebo cows wereBVDV-immunohistochemistry positive, a significantly higher incidence(P≦0.0001) versus the vaccinates. BVDV immunohistochemistry results wereeither all positive or all negative for the tissues evaluated from eachfetus.

[0089] Table 5 indicates the source of virus isolation and theprechallenge SN titers of the 2 vaccinated cows with positive BVDVisolation results. Serologic data indicates that T3 vaccinates 27 and 60both responded immunologically to vaccination. Cow 27 had a BVDV type 2SN titer at challenge that was lower than the GMT for the T3 group, butthe difference was not significant.

[0090] Intramuscular and SC vaccination collectively provided 92.9percent efficacy against fetal BVDV-2 infection, with negative BVDVisolation results occurring in fetal tissues from 13 of 14 vaccinatedcows (Table 4). This compared to a 88.9 percent rate of protection (16of 18 vaccinates were BVDV-isolation negative) in an earlier test of thesame vaccine against fetal challenge with BVDV-1 (see Example 2). Inboth of these studies, 100 percent fetal infection occurred innonvaccinated placebo cows, affirming that vaccinates were exposed to asevere challenge of immunity.

[0091] Challenge virus was isolated from the amniotic fluid of 2vaccinates, cows 27 and 60 (Table 5). As a result, both of these cowswere considered positive for BVDV infection, even though they wereviremia negative at each of 8 PC intervals. Virus isolation, as well asimmunohistochemistry methods of high specificity and sensitivity, didnot detect BVDV in the aborted fetus from cow 27, so it was of necessityreported as BVDV-negative. Abortion of this fetus due to a BVDVinfectious process in the dam was possible.

[0092] To corroborate results, two methods were used to assessprotection in cows (viremia and virus isolation from amniotic fluid) andtheir fetuses (immunohistochemistry and virus isolation in tissueculture). The most conservative result was used to determine rate ofprotection. Thus, the protection rate in vaccinated cows was consideredto be 87.5 percent (14 of 16), the percentage of cows negative for virusisolation from amniotic fluid, rather than 100 percent, the percentageof viremia-negative cows. No published BVDV challenge-of-immunity studyhas yielded 100 percent fetal protection in vaccinates against challengethat produced 100 percent fetal infection in nonvaccinated controls.Even a modified-live virus (MLV) vaccine, not commonly evaluated inpregnant cows, provided no more than 83 percent protection against fetalBVDV-1 infection in one study. (Cortese V S, Grooms, D L, Ellis J, et al(1998) Am J Vet Res. 59:1409-1413.) TABLE 1 Test groups and finalpregnancy status of cows in bovine viral diarrhea virus (BVDV) type 2fetal challenge study Termination of pregnancy No. fetuses No. cows No.cows (4) evaluated for vaccinated challenged (1) Material (3) FailedCaesarian BVDV isolation Group Treatment (Days 0, 21) (Day 119)death^(a) (2) Abortion^(b) pregnancy^(c) section (1 + 2 + 4) T1 Placebo(IM) 18 7 1 0 1 5 6 T2 Vaccine (IM) 18 8 0 4 0 4 8 T3 Vaccine (SC) 20 80 3 2 3 6

[0093] TABLE 2 Bovine viral diarrhea virus (BVDV) type 1 serologicalresponse in cows challenged with BVDV type 2 Reciprocal of BVDV type 1serum neutralizing (SN) geometric mean titer at selected test intervalsBreeding Caesarian section Treatment No. seropositive VaccinationVaccination (Days Challenge Amniocentesis of breeding^(a) group cows onday (Day 0) (Day 21) 32-41) (Day 119) (Day 147) (Days 300-301) T1 (n =7)  0/7 <2 <2 <2 <2  65.7  833.6 (n = 6)^(d) T2 (n = 8)  8/8 <2 13.7^(b)2,383.1^(c) 480.7^(c) 1,919.2^(c) 762.7 (n = 4)^(e) T3 (n = 8)  7/8(87.5%) <2 18.2^(c) 1,116.6^(c) 570.4^(c) 1,448.3^(c) 691.7 (n = 5)^(f)T2 & T3 (n = 16) 15/16 (93.8%) <2 15.8^(c) 1,631.3^(c) 523.6^(c)1,667.2^(c) 726.3 (n = 9)

[0094] TABLE 3 Bovine viral diarrhea virus (BVDV) type 2 serologicalresponse in cows challenged with BVDV type 2 Reciprocal of BVDV type 2serum neutralizing (SN) geometric mean titer at selected test intervalsTreatment No. seropositive Vaccination Vaccination Breeding ChallengeAmniocentesis Caesarian section group (no.) cows on day (Day 0) (Day 21)(Days 32-41) (Day 119) (Day 147) Dof breeding^(a) T1 (n = 7)  0/7 <2 <2<2 <2 402.6  2,823.8 (n = 6)^(g) T2 (n = 8)  8/8 <2  7.0^(b) 1,217.7^(d)285.2^(d) 2,598.6^(e)  922.2b (n = 4)^(h) T3 (n = 8)  8/8 <2 12.8^(c)1,837.6^(d) 261.7^(d) 1,217.5   621.3 (n = 5)^(i) T2 & T3 (n = 16) 16/16<2 9.5^(c) 1,495.9^(d) 273.2^(d) 1,778.7^(f)  756.9^(b) (n = 9)

[0095] TABLE 4 Summary of postchallenge cow and fetal bovine viraldiarrhea virus (BVDV) isolation results Postchallenge virus isolationmethod and incidence Fetal Treatment Anmiotic tissue immuno- Viremiafluid virus virus Fetal tissue histo group in cows^(a) isolationisolation^(b) chemistry^(b) T1 Placebo 7/7 (100%) 7/7 (100%) 6/6(100%)^(c) 6/6 (100%)^(c) (IM) T2 Vaccine 0/8 (0%)^(d) 0/8 (0%)^(d) 0/8(0%)^(d) 0/8 (0%)^(d) (IM) T3 Vaccine 0/8 (0%)^(d) 2/8 (25%)^(d) 1/6 1/6(16.7%)^(c,d) (SC) (16.7%)^(c,d) T2 & T3 0/16 (0%)^(d) 2/16 1/14(7.1%)^(d) 1/14 (7.1%)^(d) (12.5%)^(d)

[0096] TABLE 5 Maternal serum neutralization (SN) titers and results ofchallenge in cases where bovine viral diarrhea virus (BVDV) was isolatedfrom vaccinated cows or their fetuses BVDV serotype and reciprocal SNtiter at challenge Source of Test virus Termination group Cow no.isolation of pregnancy BVDV1 BVDV2 T3 27 AF Abortion 512 91 T3 60 AF,FT, IHC Caesarian section 181 152 T3 Group GMT N/A N/A 570 262

EXAMPLE 2 Materials and Methods

[0097] Animals—Fifty-nine BVDV seronegative (i.e., having serumneutralization [SN] titers <1:2) cows and heifers of breeding age andsoundness were obtained from multiple sources and maintained inisolation at research facilities in Nebraska for the duration of thestudy. Each animal was identified with duplicate ear tags, one placed ineach ear. New tags were installed in cases where an animal lost an eartag. Prior to the study, test animals were inoculated with commercialvaccines for leptospirosis, campylobacteriosis (vibriosis), andclostridial infections. Test animals were maintained under supervisionof an attending veterinarian, who clinically monitored them on a dailybasis.

[0098] Test vaccine—The test vaccine was a multivalent, modified liveinfectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) vaccine in desiccated form,rehydrated with an inactivated, liquid BVDV vaccine(CattleMaster/PregSure 5, Pfizer Inc, New York, N.Y.). The BVDVcomponent was combined with a sterile adjuvant. Potency of the BVDVimmunizing antigen was established by calculating the geometric meantiter (GMT) for 8 replicate titrations of the bulk fluid used forvaccine preparation. Following rehydration, the IBR-PI3-BRSV-BVDVvaccine was administered in 2 mL doses by either intramuscular (IM) orsubcutaneous (SC) injection. The desiccated IBR-PI3-RSV vaccinereconstituted with sterile water was used as a placebo.

[0099] Challenge virus—A noncytopathic BVDV type 1 field isolate (Strain816317, obtained from Dr. E. J. Dubovi, New York State College ofVeterinary Medicine, Cornell University) was used as a challenge agent.Virus identity was confirmed by SN and reverse transcriptase polymerasechain reaction (RT-PCR). The RT-PCR analysis was positive for BVDV type1 nucleotide sequences for the gp53 and p80 proteins and the 5untranslated region, and negative for the BVDV type 2 p125 sequence.Challenge virus potency was established at a GMT of 10^(4.3) TCID₅₀ permL by 2 replicate titrations made immediately before and afterchallenge. Challenge inoculum was given intranasally in a 4 mL divideddose, 2 mL per nostril.

[0100] Serologic assays—Serum neutralization titers for BVDV types 1 and2 were determined by a constant-virus, decreasing-serum assay in bovinecell culture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 125c.

[0101] Virus isolation—Postchallenge isolation of BVDV in bovine cellculture was attempted from peripheral cow blood, amniotic fluid, andfetal tissues. A BVDV-positive cell culture was determined by indirectimmunofluorescence using goat anti-BVDV polyclonal antibodies. Isolationof BVDV from fetal tissues was also attempted using immunohistochemistrymethods previously described. Haines D M, Clark E G, and Dubovi E J. VetPathol 1992;29:27-32. Whole blood from cows was drawn from the jugularvein in 5-10 mL samples and placed in heparin-containing tubes forpreparation of buffy coat cells used for virus isolation attempts.Amniotic fluid was collected under local anesthesia by left-flanklaparotomy and aspiration of a 3-5 mL sample from the uterus. Followingcaesarian section or spontaneous abortion, the eyes, 3 brain sections,spleen, and thymus were aseptically collected from each fetus.Supernatant from homogenized fetal tissues was used for virus isolationattempts in cell culture. For purposes of immunohistochemistryevaluation, fetal tissues were embedded in paraffin and tested induplicate using 1:800 and 1:600 ascites dilutions containing anti-BVDVmonoclonal antibodies.

[0102] Biometric data analysis—To demonstrate protection followingchallenge, a statistically significant reduction in incidence ofmaternal and fetal infection had to be demonstrated in vaccinated groups(T2 and T3) versus the placebo control group (T1). A Fisher's exact testwas used to compare incidence of cow viremia and BVDV isolation fromamniotic fluid, fetal tissue, and fetal tissue immunohistochemistry.Serum neutralization titers were analyzed using a mixed linear modelwith repeated measures. Least squares means from the analysis ofvariance were used to calculate a geometric mean titer (GMT), whichexcluded SN data for cows that were not challenged. A probability valueof P≦0.05 was used to determine statistical significance.

[0103] Fetal protection study—The 59 test animals were randomly assignedto one of three test groups, an IM placebo group (T1), an IM vaccinationgroup (T2), and a SC vaccination group (T3) as noted in Table 6. Cowswere inoculated with either vaccine or placebo on study Day 0 and Day21. In all cases, the Day 0 inoculation was administered on the leftside of the neck, and the Day 21 inoculation was administered on theright side of the neck.

[0104] On Day 32, all cows received an IM prostaglandin injection(Lutalyse, Pharmacia & Upjohn, Kalamazoo, Mich.) to synchronize estrus.Cows which displayed estrus were bred by artificial insemination withcertified BVDV-negative semen. On Day 96, at approximately 60 days ofgestation, the pregnancy status of cows was determined by rectalpalpation. On Day 103, 10 cows with confirmed pregnancies were randomlyselected from each test group, and relocated to a nearby isolationfacility (Midwest Veterinary Services, Oakland, Nebr.). On Day 117,these 30 cows were challenged by intranasal inoculation of virulentBVDV. Blood samples were collected on the day of challenge and at 8postchallenge intervals, on Days 119, 121, 123, 125, 127, 131, 138, and145, for purposes of BVDV isolation.

[0105] On Day 145 (28 days after challenge), left flank laparotomieswere performed and amniotic fluid was extracted from each cow. On Day295, approximately 7-14 days prior to anticipated calving, test cowswere transported to facilities at the University of Nebraska, Departmentof Veterinary and Biomedical Services, for caesarian section.Immediately prior to surgery, a blood sample was collected from each cowfor SN assay. Following caesarian delivery (on Days 298-300), a bloodsample was collected from each fetus. Fetuses were then euthanized andtissues were aspectically collected for purposes of BVDV isolation.

[0106] In cases where spontaneous abortions occurred, blood samples weretaken from the dam when abortion was detected and two weeks later. Thepaired blood samples and aborted fetuses were submitted for serologictesting of blood samples (University of Nebraska Veterinary DiagnositicCenter, Lincoln, Nebr.) virus isolation from fetal tissues, (PfizerCentral Research, Lincoln, Nebr.) and histopathologic evaluation offetal tissues. (Saskatoon Veterinary Biodiagnostics, Saskatoon, SK,Canada.

Results

[0107] Individual SN values for the 30 cows used in the fetal protectiontest were negative for BVDV types 1 and 2 on Day 0, confirming thatthese test animals were all immunologically naive to BVDV challenge atthe outset of the study. The GMT values (Tables 7 and 8) indicate thatIM (T2 group) and SC (T3 group) vaccination both elicited a serologicresponse following administration of two doses. All cows in the T2 andT3 groups seroconverted (SN titer >1:8) to BVDV type 1 and BVDV type 2following the second vaccine dose. At breeding (Day 34-37), the BVDVtype 1 SN titers ranged from 1:27 to 1:2,900, and the BVDV type 2 titersranged from 1:609 to 1:13,777. After vaccination, GMT values for the SCgroup were marginally higher versus the IM group at each prechallengeinterval, but the differences were not statistically significant.Twenty-eight days after challenge (Day 145), the BVDV type 1 GMT values(Table 7) showed a statistically significant difference favoring the SCvaccinates (T3 group) versus the IM (T2) group.

[0108] All cows in the placebo group (T1) remained seronegative for bothBVDV types 1 and 2 up to the time of challenge (Day 117), indicatingthat the study was not compromised by adventitious exposure. Placebocows responded serologically to challenge, but their GMT responses toBVDV types 1 and 2 on Day 145 were significantly lower than theanemnestic responses achieved by either vaccine group (Tables 7 and 8).

[0109] Between the time of amniocentesis (Day 145) and caesarian section(Day 298-300), two cows aborted, one from the T1 group and the otherfrom the T3 group. In addition, 4 bred cows (2 T1 placebo cows and onecow each from the T2 and T3 groups) were found to be not pregnant at thetime of caesarian section. These 4 cows were confirmed pregnant byrectal palpation on Day 96, approximately 60 days after breeding,indicating that unobserved abortion or fetal resorption occurred.Because fetal tissues from the 4 cows with failed pregnancies were notavailable for evaluation, these animals were removed from the study.Thus, at the conclusion of the study, 24 of the 30 cows that had beenchallenged underwent caesarian section, and a total of 26 fetusesresulting from either caesarian delivery or abortion were evaluated forBVDV isolation (Table 6).

[0110] Cow number 1317 from the T1 placebo group aborted its fetus onDay 238 (after 201 days of gestation, and 121 days after challenge).Histopathologic and bacteriologic evaluation of the fetus revealedpneumonia, necrosis of the chorionic epithelium, and Corynebacterium sp.isolated from the stomach and placenta. Paired serologic samples fromthe cow did not support IBR, BVD, or leptospirosis as the abortionetiology. Positive BVDV isolation results in cell culture were obtainedfor peripheral cow blood collected at 6, 8, and 10 days after challenge;for amniotic fluid; and for fetal brain, eye, and thymus, but not thespleen. Fetal brain, eye, thymus, and spleen were immunohistochemistrypositive for BVDV. Virus isolation and serologic evidence in this caseindicates that a BVDV infected fetus was aborted by a dam thatexperienced viremia as a result of challenge.

[0111] Cow number 1331 from the T3 vaccine group aborted its fetus onDay 249 (after 212 days of gestation, and 132 days after challenge).Histopathologic and bacteriologic evaluation of the fetus revealed adiffuse purulent pneumonia. Cultures of stomach contents and lung wereheavily overgrown with coliform bacteria. Paired post-abortion serologicsamples from the cow did not support IBR, BVD, or leptospirosis as theabortion etiology. Attempts at BVDV isolation in cell culture werenegative for cow peripheral blood collected at all 9 postchallengeintervals, and for amniotic fluid, and fetal brain, eye, spleen andthymus. Immunohistochemistry results for the fetal tissues werenegative. However, pooled fetal tissues were positive for BVDV isolationin cell culture. The conflicting results suggest the possibility ofcontamination of fetal tissues either by contact with pasture seededwith BVD challenge virus or by fomites at the necropsy facility, whereBVDV had been previously isolated. The absence of postchallenge viremiain the dam, its positive seroconversion status, and negative BVDVisolation results for specific fetal organs support the conclusion thatthis fetus was not BVDV infected as a result of challenge. Blood samplescollected from each caesarian derived fetus were assayed for SN titersto BVDV type 1 and 2. None of the 7 caesarian derived fetuses from T1placebo cows was seropositive for either BVDV type 1 or 2. Five of the17 caesarian derived fetuses from T2 and T3 vaccinates were seropositivefor BVDV type 1. Four fetuses had type 1 SN titers of either 1:2 or 1:4,and the fetus from T2 cow 1421 had a type 1 SN titer of 1:181 and a type2 SN titer of 1:512.

[0112] Postchallenge virus isolation results are shown in Table 9. Nineof 10 T1 placebo cows experienced BVDV viremia, indicating that a viablechallenge occurred. Blood samples from 19 of the 20 T2 and T3 vaccinateswere negative for BVDV viremia on each of 8 postchallenge samples. T2vaccinate 1421 was BVDV positive on Day 123, 6 days after challenge, anddelivered a fetus that was seropositive as noted above, butvirus-isolation negative. The 5 percent incidence of postchallenge BVDVviremia in T2 and T3 vaccinates was significantly less (P≦0.0001) thanthe 90 percent rate in controls.

[0113] Fetal tissues from 2 of 18 vaccinates (11.1 percent), T2 cows1301 and 1335, were positive for BVDV isolation. This compared to BVDVisolation in 8 of 8 (100 percent) fetuses from T1 placebo cows, astatistically significant difference (P≦0.0001). BVDV isolation resultswere either BVDV-positive or -negative for all fetal tissues evaluated,with one exception, T1 cow 1317, from which 3 of 4 fetal tissue sampleswere positive.

[0114] Amniotic fluid from 2 of 20 (10 percent) vaccinates was BVDVpositive, versus positive results for 10 of 10 (100 percent) of T1placebo cows, a statistically significant difference (P≦0.0001).Amniotic fluid samples from T2 vaccinates 1301 and 1335 were positive.

[0115] Immunohistochemistry results were BVDV positive for fetal tissuesfrom 2 of 18 (11.1 percent) vaccinates, T2 cows 1301 and 1335. All fetaltissues evaluated from these vaccinated cows were positive. Thiscorresponded to BVDV isolation results for the same two cows when virusisolation was attempted from amniotic fluid and from fetal tissues usingcell culture methods. All fetal tissues from 8 of 8 (100 percent) T1placebo cows were BVDV positive, a significantly higher incidence(P≦0.0001) versus the vaccinates.

[0116] Prechallenge serologic status of the three vaccinated cows withpositive BVDV isolation results is shown in Table 10. Cows 1301 and1335, which delivered BVDV-positive fetuses, and cow 1421, which wasviremic, all responded immunologically to vaccination.

[0117] Sixteen of 18 fetuses (88.9 percent) from vaccinated cows wererefractory to a challenge that produced 100 percent fetal infection innonvaccinated controls. The intranasal challenge not only mimicked thenatural route of infection, but at a dosage much greater than what wouldbe expected from field exposure. Challenge potency also exceeded thelevel that a prior study found would consistently achieve experimentalBVDV type 1 viremia and fetal infection. Ficken M, Jeevaraerathnam S,Wan Welch S K, et al: BVDV fetal infections with selected isolates. In:Proceedings of the International Symposium on Bovine Viral DiarrheaVirus, a Fifty-Year Review. Ithaca, N.Y., 1996;110-112. A noncytopathicchallenge strain was used since this is the biotype associated withpersistent infection and infection of immunotolerant fetuses. CorteseVS: Bovine virus diarrhea virus and mucosal disease. In: CurrentVeterinary Therapy 4, Food Animal Practice. Philadelphia, Pa.: WBSaunders, 1999;286-291.

[0118] Serologic data affirmed vaccine antigenicity by both IM and SCroutes of administration. All vaccinated cows seroconverted to both BVDVtypes, and the marked anemnestic response to challenge (Tables 7 and 8)resulted in GMT titers that persisted until the end of the study, 6months later. The three vaccinated cows linked to positive virusisolation also seroconverted following vaccination (Table 10).Caesarian-derived calves from seropositive cows 1301 and 1335 werepositive for BVDV. Virus isolation from seropositive cows or theirfetuses suggests humoral antibody may correlate with protection but isnot its sole determinant. Cellular or mucosal mechanisms may also beinvolved. TABLE 6 Test groups and final pregnancy status of cows inbovine viral diarrhea virus (BVDV) type 1 fetal challenge study No.fetuses evaluated No. cows No. cows Termination of pregnancy for BVDVvaccinated challenged (1) Maternal (3) Failed (4) Caesarian isolationGroup Treatment (Days 0, 21) (Days 119) death^(a) (2) Abortion^(b)pregnancy^(c) section (1 + 2 + 4) T1 Placebo (IM) 18 7 1 0 1 5 6 T2Vaccine (IM) 18 8 0 4 0 4 8 T3 Vaccine (SC) 20 8 0 3 2 3 6

[0119] TABLE 7 Bovine viral diarrhea virus (BVDV) type 1 serologicalresponse in cows challenged with BVDV type 1 Reciprocal of BVDV type 1serum neutralizing (SN) geometric mean titer at selected test intervalsCaesarian section Treatment No. seropositive Vaccination VaccinationBreeding Challenge Amniocentesis of breeding^(a) group cows on day (Day0) (Day 21) (Days 34-37) (Day 117) (Day 145) (Days 298-300) T1 (n = 10) 0/10 <2 <2 <2 <2 118.0   808.2 (n = 9)^(d) T2 (n = 10) 10/10 <2 5.5^(b)414.1^(b) 177.4^(b) 10,380.1^(b) 2,233.2^(b) (n = 10) T3 (n = 10) 10/10<2 7.3^(b) 630.2^(b) 281.2^(b)   20,169.2^(b, c) 3,804.6^(b) (n = 9)^(e)T2 & T3 (n = 20) 20/20 <2 6.3^(b) 510.8^(b) 223.3^(b) 14,469.2^(b)2,914.9^(b) (n = 19)

[0120] TABLE 8 Bovine viral diarrhea virus (BVDV) type 2 serologicalresponse in cows challenged with BVDV type 1 Reciprocal of BVDV type 2serum neutralizing (SN) geometric mean titer at selected test intervalsCaesarian section Treatment No. seropositive Vaccination VaccinationBreeding Challenge Amniocentesis of breeding^(a) group cows on day (Day0) (Day 21) (Days 34-37) (Day 117) (Day 145) (Days 298-300) T1 (n = 10) 0/10 <1.4 <1.4 <1.4 <1.4   604.9 (n = 9)^(d) T2 (n = 10) 10/10 <1.4 7.6^(b) 1,782.9^(b) 174.8^(b) 3,756.0^(b) 1,634.9^(b) (n = 10) T3 (n =10) 10/10 <1.4 17.7^(b,c) 2,749.6^(b) 309.7^(b) 4,240.4^(b) 1,879.9^(b)(n = 9)^(e) T2 & T3 (n = 20) 20/20 <1.4 11.6^(b) 2,214.1^(b) 232.7^(b)3,990.9^(b) 1,753.1^(b) (n = 19)

[0121] TABLE 9 Summary of postchallenge cow and fetal bovine viraldiarrhea virus (BVDV) isolation results Postchallenge virus isolationmethod and incidence Amniotic Fetal tissue Fetal tissue TreatmentViremia fluid virus virus immunohisto- group in cows^(a) isolationisolation^(b) chemistry^(b) T1 Placebo 9/10 (90%) 10/10 8/8 (100%)^(c)8/8 (100%)^(c) (IM) (100%) T2 Vaccine 1/10 (10%)^(d)  2/10 2/9 2/9(22.2%)^(c,d) (IM) (20%)^(d) (22.2%)^(c,d) T3 Vaccine 0/10 (0%)^(d) 0/10 (0%)^(d) 0/9 (0%)^(d) 0/9 (0%)^(c,d) (SC) T2 & T3 1/20 (5%)^(d) 2/20 2/18 2/18 (11.1%)^(d) (10%)^(d) (11.1%)^(d)

[0122] TABLE 10 Cow pre-challenge serum neutralization (SN) titers incases where bovine viral diarrhea virus (BVDV) was isolated fromvaccinated cows or their fetuses BVDV serotype and reciprocal SN titerat challenge Test Source of group Cow no. virus isolation BVDV1 BVDV2 T21301 FT, AF, IHC 128 181 T2 1335 FT, AF, IHC 109 91 T2 1421 CV 64^(a)27^(a) T2 Group GMT N/A 177.4 174.8

EXAMPLE 3

[0123] Two groups of 16 cattle were vaccinated twice subcutaneously atan interval of 3 weeks with 2 mL of L. hardjo/L. pomona combinationvaccines prepared from two adjuvant formulations: 1) 2.5% Amphigen withQuil A /cholesterol each at 250 mcg/mL, and 2) Amphigen/AI-gel. Thevaccines consisted of killed leptospires from which the culture fluidshad been removed, so free endotoxin was low. Body temperatures,injection-site reactions, and general health observations were recordedfollowing both injections. No systemic affects were seen, and localreactions were minimal and judged to be clinically acceptable. Sixteenadditional cattle were injected with saline as controls. Four weeksafter vaccination, cattle were challenged by ocular and vaginalinstillation of 5 x 10⁶ leptospires on 3 consecutive days. Half of eachtreatment group was challenged with serovar hardjo and half with pomona.Two pomona controls were eliminated from the study for unrelatedreasons, leaving 6 animals in that group. Urine collected weekly, andkidney samples collected at necropsy, 8 weeks after challenge, wereevaluated by culture, PCR, and fluorescent antibody microscopy (FA).

[0124] Following L. hardjo challenge, viable organisms were detected inurine and/or kidney cultures from 100% (8/8) of the unvaccinatedcontrols, whereas positive cultures were never obtained from anyvaccinated animal (0/16). After challenge with L. pomona, 67% (4/6) ofthe unvaccinated controls became infected on the basis of urine/kidneyculture, but none of the vaccinates was ever kidney or urine culturepositive (0/16).

[0125] Since leptospirosis is transmitted via contaminated urine, theability to prevent or reduce urinary shedding is a useful measure ofvaccine efficacy. Both vaccine formulations reduced urinary shedding ofleptospires by statistically significant amounts compared to controls.The data show a substantial benefit from vaccination with the bivalentL. hardjo/L. pomona vaccines formulated with either adjuvant;demonstrating the protection of cattle from infection with leptospiresby vaccinating with formalin-killed combination bacterins.

EXAMPLE 4 Materials and Methods

[0126] Animals—Thirty-six BVDV and Leptospira seronegative (i.e., havingBVDV serum neutralization [SN] titers <1:2 and Leptospira serovarshardjo and pomona [MAT] titers <1:20)) approximately 7-month old calveswere obtained from multiple sources and maintained in isolation atresearch facilities in Nebraska for the duration of the study. Eachanimal was identified with duplicate ear tags, one placed in each ear.New tags were installed in cases where an animal lost an ear tag. Priorto the study, test animals were vaccinated against clostridial diseasesand bovine respiratory disease agents (excluding BVD virus). Testanimals were maintained under supervision of an attending veterinarian,who clinically monitored them on a daily basis.

[0127] Test vaccines—The experimental test vaccines were liquid vaccinescontaining either formalin inactivated L. hardjo-bovis or L. pomona, orboth, and inactivated BVD type 1 and type 2 viruses. The BVDV componentswere combined with a sterile adjuvant. Potency of the BVDV immunizingantigens was established by calculating the geometric mean titer (GMT)for 8 replicate titrations of the bulk fluid used for vaccinepreparation. Potency of the Leptospira immunizing antigens wasestablished in accordance with a hamster lethality model procedure. Theadjuvants in the experimental test vaccines were comprised of either2.5% Amphigen (v/v) with Quil A/cholesterol each at 100 mcg/ml, with orwithout 2% (v/v) aluminum hydroxide; 2.5% Amphigen (v/v) with QuilA/Dimethyl dioctadecylammonium bromide (DDA) each at 100 mcg/ml, with orwithout 2% (v/v) aluminum hydroxide. The experimental test vaccines wereadministered in 5 mL dose by subcutaneous (SC) injection. A monovalentL. hardjo-bovis bacterin was used as a positive control permanufacturer's instructions. A placebo vaccine containing physiologicalsaline was used as a negative control.

[0128] Challenge bacteria—Leptospira borgpetersenii serovar hardjo typehardjo-bovis strain 203 (National Animal Disease Center, Ames, Iowa),was used as the challenge agent. L. hardjo-bovis challenge material wasprepared as first passage organisms which had been isolated from theurine of cattle experimentally infected with L. hardjo-bovis. Thechallenge material was administered once daily for three consecutivedays. Each challenge day, a total of two mL of challenge material,containing approximately 2.5×10⁶ L. hardjo-bovis organisms/mL, wasadministered across three separate anatomical sites. The route ofchallenge was instillation into the conjunctival sac of each eye (½ mLeach) and into the vagina (1 mL).

[0129] Serologic assays—Serum neutralization titers for BVDV types 1 and2 were determined by a constant-virus, decreasing-serum assay in bovinecell culture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 1 25c. Serum microscopic agglutinationtiters (MAT) for L. hardjo-bovis and L. pomona were conducted using astandard test at a qualified veterinary diagnostic center (CornellUniversity College of Veterinary Medicine Diagnostic Laboratory).

[0130] Leptospira isolation—Urine samples and kidney tissue homogenates(pooled left and right kidney) were examined for the presence ofLeptospira. Urine and kidney cultures were examined for Leptospira onceweekly for up to 8 weeks using standard procedures. Leptospirafluorescent antibody (FA) techniques were conducted using a standardtest at a qualified veterinary diagnostic center (Cornell UniversityCollege of Veterinary Medicine Diagnostic Laboratory).

[0131] Biometric data analysis—To demonstrate protection followingchallenge, a statistically significant reduction in incidence ofLeptospira infection had to be demonstrated in vaccinated groups (Table11)(T02, T03, T04 and T05) versus the placebo control group (T1). Datafor kidney colonization and urinary shedding were summarized bytreatment and timepoint. Comparisons between treatments were made as tothe percent of animals with Leptospira detected in the kidney.Comparisons between treatments were made as to the percent of animalswith Leptospira detected in the urine. A Fisher's Exact test was usedfor the analyses above. Duration of Leptospira shedding in the urine wasalso compared using a general linear mixed model. A probability value ofP≦0.05 was used to determine statistical significance.

[0132] Leptospira protection study-The 36 test animals were randomlyassigned to one of six test groups as indicated in Table 11. On Day 0and Day 21, each animal assigned to T01-T05 received one 5 mL SC dose ofthe appropriate experimental test or placebo vaccine. On Day 0 and Day28, each animal assigned to T06 received one 2 mL SC dose of thepositive control vaccine. On Days 57-59, all animals were challengedwith L. hardjo-bovis strain 203 as outlined above.

[0133] Blood samples were collected from each animal on Days 0, 21,35,56, 84, and 111 for determination of BVDV type 1 and type 2 titers.

[0134] Urine samples (approximately 45 mL) were collected from eachanimal on Days—1, 56, 70, 77, 84, 91, 98 and 105 for leptospireisolation as described above.

[0135] Animals were euthanized on Days 112 and 113 and kidneys evaluatedfor the presence of leptospires as described above.

Results

[0136] The GMT values (Table 12) indicate that all the animals receivingBVDV-Leptopsira combination vaccines (T02, T03, T04 and T05) elicited aserologic response following administration of two vaccine doses. Allthe animals in groups T02, T03, T04 and T05 seroconverted (SN titer≧1:8) to BVDV type 1 following the second vaccine dose. All the animalsin groups T02, T04 and T05 seroconverted (SN titer ≧1:8) to BVDV type 2following the second vaccine dose. All cows in the placebo group (T01)or in the group that received monovalent L. hardjo-bovis vaccineremained seronegative from both BVDV types 1 and 2, indicating that thestudy was not compromised by adventitious exposure. Collectively, theBVDV serology data shows for the first time that combination vaccinescomprising inactivated BVDV types 1 and 2 and inactivated L. hardjobovisand L. pomona formulated in four different adjuvants can induce aprotective response against BVDV disease in cattle, since a cow BVDV SNtiter of ≧1:8 is known in the art to be indicative of protection againstBVDV disease.

[0137] The Leptospira urine and kidney results (Table 13) indicate thatall animals that received BVDV-Leptospira combination vaccines (T02,T03, T04 and T05) were urine culture (CX) negative (Table 13, column2)at all eight timepoints tested and kidney culture negative (Table 13,column 8) at necropsy (Day 112 or 113). Cows that received Leptospiramonovalent vaccine (T06, positive control) were similarly protectedagainst Leptospira infection. In contrast, cows that received placebovaccine (T01, negative control) were infected based on urine (Table 13,column 2) and kidney culture (Table 13, column 8), indicating thevaccine challenge study was valid. Collectively, the L. hardjobovisisolation data shows for the first time that combination vaccinescomprising inactivated BVDV types 1 and 2 and inactivated L. hardjobovisand L. Pomona formulated in four different adjuvants can induce aprotective response against Leptospira disease in cattle. TABLE 11 Testgroups of calves in BVDV-Leptospira combination vaccine study NumberRoute of Number of Dosing of Dose Administra- Doses/ Treatment VaccineAnimals Volume tion Animal Interval T01 Saline placebo 6 5 mL SC 2 3weeks T02 L. hardjobovis- 6 5 mL SC 2 3 weeks L. pomona-BVDV-1- BVDV-2in QAC T03 L. hardjobovis- 6 5 mL SC 2 3 weeks L. pomona-BVDV-1- BVDV-2in QAC/AIOH T04 L. hardjobovis- 6 5 mL SC 2 3 weeks L. pomona-BVDV-1-BVDV-2 in DDA T05 L. hardjobovis- 6 5 mL SC 2 3 weeks L. pomona-BVDV-1-BVDV-2 in DDA/AIOH T06 L. hardjobovis 6 2 mL SC 2 4 weeks monovalent

[0138] TABLE 12 BVDV Types 1 and 2 Serum Neutralization ReciprocalGeometric Mean Titers and Ranges (#-#) on Day 35 Serum VirusNeutralization Reciprocal Geometric Mean Titer and Range (#-#) on Day 35Treatment BVD Virus Type 1 BVD Virus Type 2 T01 <2(<2-<2) <2(<2-<2) T021084.9 (609-2435) 20.8 (16-54) T03  148.0 (<2-1218)  5.8 (<2-19) T041877.9 (1024-2896) 34.0 (19-91) T05 1084.6 (152-3444) 36.1 (10-91) T06<2(<2-<2) <2(<2-<2)

[0139] TABLE 13 Efficacy Results of BVDV-Leptospira Combination VaccinesAgainst Leptospira hardjo-bovis Challenge Percent of Calves LeastSquares Mean Percent of Calves Ever Positive for Percent Days of EverPositive Leptospira Leptospira for Leptospira in in Urine Positive UrineKidneys Treatment CX FA PCR CX FA PCR CX FA PCR T01 n = 6 100^(a)83.3^(a) 83.3^(a) 50.9^(a) 29.1^(a) 37.1^(a) 83.3^(a)   0^(a) 16.7^(a)T02 n = 6  0^(b)   0^(b)   0^(b)   0^(b)   0^(b)   0^(b)   0^(b)   0^(a)  0^(a) T03 n = 6  0^(b)   0^(b)   0^(b)   0^(b)   0^(b)   0^(b)   0^(b)50.0^(a)   0^(a) T04 n = 6  0^(b)   0^(b)   0^(b)   0^(b)   0^(b)  0^(b)   0^(b)   0^(a)   0^(a) T05 n = 6  0^(b)  16.7^(ab)  16.7^(ab)  0^(b)  0.3^(b)  0.4^(b)   0^(b) 16.7^(a)   0^(a) T06 n = 6  0^(b)  0^(b)  33.3^(ab)   0^(b)   0^(b)  1.6^(b)   0^(b)   0^(a)   0^(a)

EXAMPLE 5 Materials and Methods

[0140] Animals—Twenty BVDV seronegative (i.e., having serumneutralization [SN] titers <1:2) cows were obtained and maintained inresearch isolation facilities for the duration of the study. Each animalwas identified with duplicate ear tags, one placed in each ear. New tagswere installed in cases where an animal lost an ear tag. Test animalswere maintained under supervision of an attending veterinarian, whoclinically monitored them on a daily basis.

[0141] Test vaccine—The test vaccine was a multivalent, modified liveinfectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) vaccine in desiccated form,rehydrated with an inactivated, liquid 8-way BVDV-Leptospiraspp-Campylobacter fetus containing vaccine in a QuilA/cholesterol/Amphigen adjuvant. (Pfizer Inc, New York, N.Y.) The liquidcomposition consisted of inactivated BVDV-1 and -2 viruses, fiveinactivated Leptospira species (L. canicola, L. grippotyphosa, L.borgpetersenii hardjo-prajitno, L. icterohaemorrhagiae and L.interrogans pomona) and inactivated C. fetus bacterin combined with aQuil A/cholesterol/Amphigen adjuvant sterile adjuvant. Control vaccineconsisted of the five inactivated Leptospira spp. described above andinactivated C. fetus bacterin combined with a sterile mineral oil(Drakeol) adjuvant. Test vaccine was given by subcutaneous injection andcontrol vaccine was given by intramuscular injections. Vaccines wereadministered on Day 0 on the right side of the neck and on Day 21 on theleft side of the neck.

[0142] Challenge virus—A cytopathic BVDV type 2 field isolate (Strain24515) was used as a challenge agent. Challenge virus potency wasestablished at a GMT of 10^(5.4) TCID₅₀/5 mL by 2 replicate titrationsmade immediately before and after challenge. Challenge inoculum wasgiven intranasally on Day 42 in a 5 mL divided dose, approximately 2.5mLs per nostril.

[0143] Serologic assays—Serum neutralization titers for BVDV types 1 and2, BHV-1, P13 and BRSV were determined by a constant-virus,decreasing-serum assay in bovine cell culture using standard procedures.C. fetus antibody titers were determined by a standard agglutinationassay.

[0144] Virus isolation—Postchallenge (PC) isolation of BVDV in bovinecell culture was conducted from peripheral white blood cells (buffycoats)on Days 42 and 45-52. A BVDV-positive cell culture was determinedby indirect immunofluorescence using goat anti-BVDV polyclonalantibodies. Whole blood from cows was drawn from the jugular vein in5-10 mL samples and placed in heparin-containing tubes for preparationof buffy coat cells used for virus isolation attempts.

[0145] Clinical Disease Scoring—Each animal was scored on Days 40-56post-challenge. A normal animal with no clinical signs received a scoreof zero. An animal with nonspecific clinical signs (eg nasal discharge,abnormal respiration, and lethargy) not specific for acute BVD virusinfection received a score of one. A score of two was assigned to anyanimal with acute BVD clinical disease in which clinical signs as awhole were moderate and specific for acute BVD virus infection. Clinicalsigns include nasal discharge, abnormal respiration, lethargy,gauntness, ocular discharge, hypersalivation, diarrhea, dehydration,lameness and/or reluctance to move. An animal with clinical signs thatas a whole were severe in degree was assigned a score of three.

[0146] Biometric data analysis—For serum virus neutralization, titerswere transformed to log base 2 and analyzed by a mixed linear model withrepeated measures. Backtransformation was done to calculate geometricmean titer (GMT). Percent number of days with positive virus isolationwas anluzed using a mixed linear model. Pairwise comparison of testversus control vaccine groups were made. A probability value of P<0.05was used to determine statistical significance.

Results

[0147] No adverse events were observed during or immediately followingadministration of the 2 vaccine doses.

[0148] All cows were seronegative to BVDV types 1 and 2 and BHV-1 priorto vaccination (Day 0), confirming that the test animals wereimmunologically naive to BVDV and BHV-1 at the outset of the study. TheGMT values for all five viral fractions on Day 0 and Day 35 (14 dayspost-second vaccination) are shown in Table 14. TABLE 14 Serum viralneutralization titers to BVDV type 1, BVDV type 2, BHV-1, PI3 and BRSVand agglutination antibody titers to C. fetus prior to (Day 0) andfollowing vaccine administration. Vaccine BVDV-1 BVDV-2 BHV-1 PI3 BRSVC. fetus Vaccine Components 0 35 0 35 0 35 0 35 0 35 0 35 Control 5Leptospira 1 1^(a) 1  1^(a) 1  1^(a) 140 175  8 14 57 260 spp., C. fetusTest BHV-1, PI3 1 8^(b) 1 18^(b) 1 145^(b) 155 453 16 57 26 422 BRSV,BVDV-1 BVDV-2 5 Leptospira spp., C. fetus

[0149] Results show that the 11-way test vaccine composition wasimmunogenic in cattle since differences in antibody titers to all 5viruses were observed between pre-vaccination (Day 0) andpost-vaccination (Day 35) timepoints. In addition, post-vaccination (Day35) titers to C. fetus were similar between the 5-way control and 11-waytest vaccine, demonstrating that the presence of the modified-live andkilled viral fractions in the 11-way vaccine did not interfere withability of the host to mount an immune response against the C. fetusbacterial fraction.

EXAMPLE 6 Materials and Methods

[0150] Animals—Thirty male and female calves were selected for the studyfrom a single herd. The ages of these calves were estimated to be 6 to 8months based on their body weights on Day 0. An additional ten calvesfrom the same herd [six were not] were enrolled on Day 18; however, theyhad not been weighed. Prior to being enrolled in the study (Day 0 forT02, T03, T04, and Day 18 for T01) all animals were seronegative (SVN<1:2) for antibodies to BVD virus Type 1 and Type 2. Each animal wasidentified with duplicate ear tags, one placed in each ear. New tagswere installed in cases where an animal lost an ear tag. Test animalswere maintained under supervision of an attending veterinarian, whoclinically monitored them on a daily basis.

[0151] Test vaccine—The test vaccines were prepared by reconstitutingthe lyophilized modified live virus vaccine CattleMaster™, containingmodified live infectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) with one of the killed BVDliquid diluents (containing BVDV types 1 and 2) prepared in amicrofluidized saponin-based (Quil A or GPI-0100) oil-in-water emulsion.The method for preparation of a microfluidized saponin-basedoil-in-water emulsion is described in application Ser. No. 60/460,301,filed Apr. 4, 2003, incorporated herein by reference. (Treatment groupsare shown in Table 15). TABLE 15 Treatment Groups used in Saponin-basedOil-in-Water Emulsion Vaccine Efficacy Study # # of Group of calvesVaccine Fractions Saponin Adjuvant Doses T01 10 0.9% saline none 2 T0210 Modified-live BHV-1 Quil A Modified-live PI3 Modified-live BRSV 0.5mg/dose 2 Killed BVDV 1 Killed BVDV 2 T03  5 Modified-live BHV-1GPI-0100 Modified-live PI3 Modified-live BRSV 0.5 mg/dose 2 Killed BVDV1 Killed BVDV 2 T03 15 Modified-live BHV-1 GPI-0100 Modified-live PI3Modified-live BRSV 1.0 mg/dose 2 Killed BVDV 1 Killed BVDV 2

[0152] Vaccines were administered as a single 2 mL dose subcutaneously(SC) on the right side of the neck for the first administration (Day 0and/or Day 2) and on the left side of the neck for the secondadministration (Day 21). Injections were administered in the lateralneck approximately midway between the scapula and the poll.

[0153] Challenge virus—The challenge virus was non-cytopathic BovineViral Diarrhea virus (BVDV) Type 2, strain 24515. On Day 42 a 5 mL doseof the challenge virus preparation (approximately 2.5 mL per nostril)was administered intranasally (needle-less syringe administration) toanimals in treatments T01, T02, T03 and T04. The challenge material wastitrated for virus content (two replicates per assay) prior to andfollowing challenge administration. The mean titers pre-challenge andpost-challenge were 5.5 log₁₀ and 5.3 log₁₀ per 5 mL dose, respectively.

[0154] Serologic assays—Blood samples (two 13 mL SST tubes) for BVDserology were collected o Days 0, 21, 35, 43, and 57. Serumneutralization titers for BVDV types 1 and 2, BHV-1, were determined bya constant-virus, decreasing-serum assay in bovine cell culture usingstandard procedures.

[0155] Total White Blood Cell (WBC) Counts: Blood samples (One 4 mL EDTAtube) for total WBC determination were collected from T01-T04 animals onDays 41, 42 and 43 (prior to challenge and following challenge) and onDays 44 through 57. Blood samples were processed and transferred toPhysicians Laboratory Services, Inc. for analysis. The results weretransferred electronically into a Clinical Data Management System

[0156] Virus isolation—Blood samples (one 8 mL CPT tube) for BVD virusisolation were collected from T01-T04 on Day 43 (prior to challenge) andon Days 44-57. A BVDV-positive cell culture was determined by indirectimmunofluorescence using goat anti-BVDV polyclonal antibodies

[0157] Clinical Disease Scoring—Clinical disease scores of 0, 1, 2, or 3based on clinical signs attributable to BVD 2 infection (see above,example 4), were made for each animal T01-T04 on Days 41 through 43(prior to challenge) and Days 44 through 57.

[0158] Biometric data analysis—For serum virus neutralization, titerswere transformed to log base 2 and analyzed by a mixed linear model withrepeated measures. Backtransformation was done to calculate geometricmean titer (GMT). Percent number of days with positive virus isolationwas analyzed using a mixed linear model. Pairwise comparison of testversus control vaccine groups were made. A probability value of P≦0.05was used to determine statistical significance.

Results

[0159] No untoward systemic reactions were observed in any of theanimals during the vaccination phase of the study (Days 0 through 42).

[0160] The geometric mean reciprocal SVN titers for antibodies to theBVD virus Type 1 and Type 2 are summarized in Tables 16 and 17. TABLE 16Geometric Mean SVN Titers for Antibodies to BVD Virus Type 1 BVD-1Geometric Mean Reciprocal SVN Titers on Study Day Treatment N 0 21 3543¹ 57 T01 saline 10 NS¹ <2^(b)  <2^(b)  <2^(b)   43.6^(b) T02 Quil A,10 <2 12.5^(a) 2393.6^(a) 2797.9^(a) 31651.6^(a) 0.5 mg T03 GPI, 5 <222.1^(a, c) 8480.8^(c) 7912.9^(c) 92682.0^(a) 0.5 mg T04 GPI, 15 <224.6^(a, c) 6968.7^(c) 6136.9^(c) 61857.7^(a) 1.0 mg

[0161] TABLE 17 Geometric Mean SVN Titers for Antibodies to BVD VirusType 2 BVD-2 Geometric Mean Reciprocal SVN Titers on Study Day TreatmentN 0 21 35 43² 57 T01 saline 10 NS¹ <2^(b)  <2^(b)  <2^(b)  494.6^(b) T02Quil A, 10 <2  4.0^(a) 469.4^(a) 587.1^(a) 75281.2^(a) 0.5 mg T03 GPI, 5<2 <2^(b) 100.3^(c)  87.4^(c) 18820.1^(c) 0.5 mg T04 GPI, 15 <2 <2^(b)125.5^(b) 100.1^(c) 18604.0^(c) 1.0 mg

[0162] As shown in Tables 16 and 17, all three saponin containing oil-inwater emulsion adjuvants induced statistically significant antibodytiters to BVDV type 1 and type 2 viruses on Days 21, 35, 43, and 57.Collectively, these data demonstrate that vaccine compositionscomprising modified-live BHV-1, PI3, BRSV and at least one additionalantigen and an adjuvant that comprises a saponin containing oil-in-wateremulsion is immunogenic in cattle. In addition, these data demonstratesuch vaccines comprising saponin containing oil-in-water microfluidizedemulsion as the adjuvant are immunogenic in cattle.

What is claimed is:
 1. An immunogenic composition comprising: a modifiedlive Bovine Herpes Virus (BHV-1); a modified live parainfluenza virusType 3 (PI3); a modified live Bovine Respiratory Syncytial Virus (BRSV);an adjuvant; at least one antigen;and a veterinary-acceptable carrier.2. The immunogenic composition of claim 1, wherein said antigen isinactivated.
 3. The immunogenic composition of claim 1, wherein saidadjuvant comprises a saponin.
 4. The immunogenic composition of claim 3,wherein said adjuvant comprises saponin containing oil-in-wateremulsion.
 5. The immunogenic composition of claim 4, wherein saidadjuvant comprises Quil A, Amphigen and cholesterol.
 6. The immunogeniccomposition of claim 5, wherein said Quil A, Amphigen and cholesteroloil-in-water emulsion is microfluidized.
 7. The immunogenic compositionof claim 1, wherein said antigen comprises at least one antigen selectedfrom the group consisting of Bovine Viral Diarrhea Virus (BVDV-1),Bovine Viral Diarrhea Virus (BVDV-2), Leptospira canicola, Leptospiragrippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospiraicterohaemmorrhagia, Leptospira interrogans pomona, Leptospiraborgpetersenii hardjo-bovis, and Campylobacter fetus.
 8. The immunogeniccomposition of claim 7, wherein said Bovine Viral Diarrhea Virus(BVDV-1) is cytopathic.
 9. The immunogenic composition of claim 7,wherein said Bovine Viral Diarrhea Virus (BVDV-1) is noncytopathic. 10.The immunogenic composition of claim 7, wherein said Bovine ViralDiarrhea Virus (BVDV-2) is cytopathic.
 11. The immunogenic compositionof claim 7, wherein said Bovine Viral Diarrhea Virus (BVDV-2) isnoncytopathic.
 12. A method of inducing an immune response againstBovine Herpes Virus Type 1 in an animal subject, comprisingadministering an immunologically effective amount of the composition ofclaim 1 and a veterinarly-acceptable carrier.
 13. A method of inducingan immune response against Bovine Viral Diarrhea Virus Type-1 in ananimal subject, comprising administering an immunologically effectiveamount of the composition of claim 1 and a veterinary-acceptablecarrier.
 14. A method of inducing an immune response against BovineViral Diarrhea Virus Type-2 in an animal subject, comprisingadministering an immunologically effective amount of the composition ofclaim 1 and a veterinary-acceptable carrier.
 15. A method of inducing animmune response against parainfluenza virus Type 3 (PI3) in an animalsubject, comprising administering an immunologically effective amount ofthe composition of claim 1 and a veterinary-acceptable carrier.
 16. Amethod of inducing an immune response against Bovine RespiratorySyncytial Virus (BRSV) in an animal subject, comprising administering animmunologically effective amount of the composition of claim 1 and aveterinary-acceptable carrier.
 17. A method of inducing an immuneresponse against Campylobacter fetus in an animal subject, comprisingadministering an immunologically effective amount of the composition ofclaim 1 and a veterinary-acceptable carrier.
 18. A method of inducing animmune response against an antigen selected from the group consisting ofLeptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interroganspomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava,Neospora caninum, Trichomonus fetus, Mycoplasma, bovis, Haemophilussomnus, Mannheimia haemolytica and Pasturella multocida in an animalsubject, comprising administering an immunologically effective amount ofthe composition of claim 1 and a veterinary-acceptable carrier.
 19. Themethod of any one of claims 12-18, wherein said immune response is acellular or humoral immune response.
 20. A vaccine compositioncomprising: a modified live Bovine Herpes Virus (BHV-1); a modified liveparainfluenza virus Type 3 (PI3); a modified live Bovine RespiratorySyncytial Virus (BRSV); an adjuvant; at least one antigen; and aveterinarly-acceptable carrier.
 21. The vaccine composition of claim 20,wherein said antigen is inactivated.
 22. The vaccine composition ofclaim 20, wherein said ajduvant comprises a saponin.
 23. The vaccinecomposition of claim 20, wherein said adjuvant comprises saponincontaining oil-in-water emulsion.
 24. The vaccine composition of claim23, wherein said saponin containing oil-in-water emulsion ismicrofludized.
 25. The vaccine composition of claim 20, wherein saidadjuvant comprises Quil A, Amphigen and cholesterol.
 26. The vaccinecomposition of claim 25, wherein said Quil A, Amphigen and cholesterol,oil-in-water emulsion is microfludized.
 27. The vaccine composition ofclaim 20, wherein said antigen comprises at least one antigen selectedfrom the group consisting of Bovine Viral Diarrhea Virus (BVDV-1),Bovine Viral Diarrhea Virus (BVDV-2), Leptospira canicola, Leptospiragrippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospiraicterohaemmorrhagia, Leptospira interrogans pomona, Leptospiraborgpetersenii hardjo-bovis and Campylobacter fetus.
 28. The vaccinecomposition of claim 27, wherein said Bovine Viral Diarrhea Virus(BVDV-1) is cytopathic.
 29. The vaccine composition of claim 27, whereinsaid Bovine Viral Diarrhea Virus (BVDV-1) is noncytopathic.
 30. Thevaccine composition of claim 27, wherein said Bovine Viral DiarrheaVirus (BVDV-2) is cytopathic.
 31. The vaccine composition of claim 27,wherein said Bovine Viral Diarrhea Virus (BVDV-2) is noncytopathic. 32.A method of preventing abortion caused by a virus selected from thegroup consisting of BHV-1 in an animal comprising administering to saidanimal a therapeutically effective amount of the vaccine composition ofclaim
 20. 33. The method of claim 32, wherein said animal is a cow, acalf, a heifer, a steer or a bull.
 34. The method of claim 33, whereinsaid animal is a lactating cow.
 35. The method of claim 33, wherein saidanimal is a pregnant cow.
 36. The method of claim 33, wherein saidanimal is a prebreeding cow or heifer.
 37. The method of claim 32,wherein said vaccine is administered intramuscularly.
 38. The method ofclaim 32, wherein said vaccine is administered subcutaneously.
 39. Themethod of claim 32, wherein said vaccine contains from about 10³ toabout 10¹⁰ colony forming units per dose of each virus.
 40. The methodof claim 32, wherein the amount of said vaccine administered is fromabout 0.5 to about 5.0 ml per dose.
 41. The method of claim 32, whereinthe amount of said vaccine administered is about 5 ml per dose.
 42. Themethod of claim 32, wherein the amount of said vaccine administered isabout 2 ml per dose.
 43. A method of treating or preventing a disease ordisorder in an animal caused by infection with a virus selected from thegroup consisting of BVDV Type 1 or Type 2, BHV-1, PI3 or BRSV comprisingadministering to said animal a therapeutically effective amount of thevaccine composition of claim
 20. 44. The method of claim 43, whereinsaid animal is a cow, a calf, a heifer, a steer or a bull.
 45. Themethod of claim 44, wherein said animal is a lactating cow.
 46. Themethod of claim 44, wherein said animal is a prebreeding cow or heifer.47. The method of claim 44, wherein said animal is a pregnant cow. 48.The method of claim 43, wherein said vaccine is administeredintramuscularly.
 49. The method of claim 43, wherein said vaccine isadministered subcutaneously.
 50. The method of claim 43, wherein saidvaccine contains from about 10³to about 10¹⁰ colony forming units perdose.
 51. The method of claim 43, wherein the amount of said vaccineadministered is from about 0.5 to about 5.0 ml per dose.
 52. The methodof claim 51, wherein the amount of said vaccine administered is about 5ml per dose.
 53. The method of claim 51, wherein the amount of saidvaccine adminstered is about 2 ml per dose.
 54. A method of treating orpreventing a disease or disorder in an animal caused by infection withan antigen selected from the group consisting Leptospira canicola,Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno,Leptospira icterohaemmorrhagia, Leptospira interrogans pomona,Leptospira borgpetersenii hardjo-bovis, Leptospira Bratislava,Campylobacter fetus, Neospora caninum, Trichomonus fetus, Mycoplasma,bovis, Haemophilus somnus, Mannheimia haemolytica and Pasturellamultocida, comprising administering to said animal a therapeuticallyeffective amount of the vaccine composition of claim
 20. 55. The methodof claim 54, wherein said animal is a cow, a calf, a heifer, a steer ora bull.
 56. The method of claim 55, wherein said animal is a lactatingcow.
 57. The method of claim 55, wherein said animal is a pregnant cow.58. The method of claim 55, wherein said animal is a prebreeding cow orheifer.
 59. The method of claim 54, wherein said vaccine is administeredintramuscularly.
 60. The method of claim 54, wherein said vaccine isadministered subcutaneously.
 61. The method of claim 54, wherein saidvaccine contains from about 10³ to about 10¹⁰ colony forming units perdose of each virus.
 62. The method of claim 54, wherein the amount ofsaid vaccine administered is from about 0.5 to about 5.0 ml per dose.63. The method of claim 62, wherein the amount of said vaccineadministered is about 5 ml per dose.
 64. The method of claim 62, whereinthe amount of said vaccine administered is about 2 ml per dose.
 65. Amethod of preventing persistent fetal infection in an animal subject,comprising administering to said animal an effective amount of thevaccine composition of claim
 20. 66. The method of claim 65, whereinsaid animal is a cow, a calf, a heifer, a steer or a bull.
 67. Themethod of claim 66, wherein said animal is a lactating cow.
 68. Themethod of claim 66, wherein said animal is a pregnant cow.
 69. Themethod of claim 66, wherein said animal is a prebreeding cow or heifer.70. The method of claim 66, wherein said vaccine is administeredintramuscularly.
 71. The method of claim 66, wherein said vaccine isadministered subcutaneously.
 72. The method of claim 66, wherein saidvaccine contains from about 10³ to about 10¹⁰ colony forming units perdose of each virus.
 73. The method of claim 65, wherein the amount ofsaid vaccine administered is from about 0.5 to about 5.0 ml per dose.74. The method of claim 73, wherein the amount of said vaccineadministered is about 5 ml per dose.
 75. The method of claim 73, whereinthe amount of said vaccine administered is about 2 ml per dose.
 76. Avaccine composition comprising: a modified live Bovine Herpes Virus(BHV-1); a modified live parainfluenza virus Type 3 (PI3); a modifiedlive Bovine Respiratory Syncytial Virus (BRSV); a cytopathic BVD-2; aBVD-1; an adjuvant; at least one antigen selected from the groupconsisting of Leptospira canicola, Leptospira grippotyphosa, Leptospiraborgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia, andLeptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis,Leptospira Bratislava and Campylobacter fetus; and aveterinary-acceptable carrier.
 77. The vaccine composition of claim 76,wherein said adjuvant comprises saponin containing oil-in-wateremulsion.
 78. The vaccine composition of claim 76, wherein said saponincontaining oil-in-water emulsion is microfluidized.
 79. The vaccinecomposition of claim 76, wherein said adjuvant comprises Quil A,Amphigen and cholesterol.
 80. The vaccine composition of claim 76,wherein said cytopathic BVD-2 is inactivated.
 81. The vaccinecomposition of claim 76, wherein said BVD-1 is cytopathic.
 82. Thevaccine composition of claim 76, wherein said cytopathic BVD-1 isinactivated.
 83. The immunogenic composition of claim 4, wherein saidsaponin containing oil-in-water emulsion is microfluidized.